Notch binding agents and antagonists and methods of use thereof

ABSTRACT

The present invention relates to Notch-binding agents and Notch antagonists and methods of using the agents and/or antagonists for treating diseases such as cancer. The present invention provides antibodies that specifically bind to a non-ligand binding region of the extracellular domain of one or more human Notch receptor, such as Notch2 and/or Notch3, and inhibit tumor growth. The present invention further provides methods of treating cancer, the methods comprising administering a therapeutically effective amount of an antibody that specifically binds to a non-ligand binding region of the extracellular domain of a human Notch receptor protein and inhibits tumor growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/782,671, filed Mar. 1, 2013, which is a divisional of U.S.application Ser. No. 13/472,305, filed May 15, 2012, which is adivisional of U.S. application Ser. No. 12/499,627, filed Jul. 8, 2009,now U.S. Pat. No. 8,226,943, which claims the priority benefit of U.S.Provisional Application No. 61/112,699, filed Nov. 7, 2008, U.S.Provisional Application No. 61/112,701, filed Nov. 7, 2008, and U.S.Provisional Application No. 61/079,095, filed Jul. 8, 2008, each ofwhich is hereby incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name:sequencelisting.ascii.txt; Size: 141 kilobytes; and Date of Creation:Mar. 12, 2013) filed herewith is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions comprising an agent thatbinds a human Notch receptor and methods of using those compositions forthe treatment of cancer and other diseases. More specifically, thepresent invention provides, for example, antibodies that specificallybind to a non-ligand binding region of the extracellular domain of ahuman Notch receptor and inhibit tumor growth. The present inventionfurther provides methods of treating cancer, the method comprisingadministering a therapeutically effective amount of an antibody thatspecifically binds to a non-ligand binding region of the extracellulardomain of a human Notch receptor protein and inhibits tumor growth.

2. Background

The Notch signaling pathway is one of several critical regulators ofembryonic pattern formation, post-embryonic tissue maintenance, and stemcell biology. More specifically, Notch signaling is involved in theprocess of lateral inhibition between adjacent cell fates and plays animportant role in cell fate determination during asymmetric celldivisions. Unregulated Notch signaling is associated with numerous humancancers where it can alter the developmental fate of tumor cells tomaintain them in an undifferentiated and proliferative state (Brennanand Brown, 2003, Breast Cancer Res. 5:69). Thus carcinogenesis canproceed by usurping homeostatic mechanisms controlling normaldevelopment and tissue repair by stem cell populations (Beachy et al.,2004, Nature 432:324).

The Notch receptor was first identified in Drosophila mutants withhaploinsufficiency resulting in notches at the wing margin, whereasloss-of-function produces an embryonic lethal “neurogenic” phenotypewhere cells of the epidermis switch fate to neural tissue (Moohr, 1919,Genet. 4:252; Poulson, 1937, PNAS 23:133; Poulson, 1940, J. Exp. Zool.83:271). The Notch receptor is a single-pass transmembrane receptorcontaining numerous tandem epidermal growth factor (EGF)-like repeatsand three cysteine-rich Notch/LIN-12 repeats within a largeextracellular domain (Wharton et al., 1985, Cell 43:567; Kidd et al.,1986, Mol. Cell. Biol. 6:3094; reviewed in Artavanis et al., 1999,Science 284:770). Four mammalian Notch proteins have been identified(Notch1, Notch2, Notch3, and Notch4), and mutations in these receptorsinvariably result in developmental abnormalities and human pathologiesincluding several cancers as described in detail below (Gridley, 1997,Mol. Cell Neurosci. 9:103; Joutel & Tournier-Lasserve, 1998, Semin. CellDev. Biol. 9:619-25).

Notch receptors are activated by single-pass transmembrane ligands ofthe Delta, Serrated, Lag-2 (DSL) family. There are five known Notchligands in mammals: Delta-like 1 (DLL1), Delta-like 3 (DLL3), Delta-like4 (DLL4), Jagged 1 (JAG1) and Jagged 2 (JAG2) characterized by a DSLdomain and tandem EGF-like repeats within the extracellular domain. Theextracellular domain of the Notch receptor interacts with that of itsligands, typically on adjacent cells, resulting in two proteolyticcleavages of Notch, one extracellular cleavage mediated by an ADAM (ADisintegrin And Metallopeptidase) protease and one cleavage within thetransmembrane domain mediated by gamma secretase. This latter cleavagegenerates the Notch intracellular domain (ICD), which then enters thenucleus where it activates the CBF1, Suppressor of Hairless [Su(H)],Lag-2 (CSL) family of transcription factors as the major downstreameffectors to increase transcription of nuclear basic helix-loop-helixtranscription factors of the Hairy and Enhancer of Split [E(spl)] family(Artavanis et al., 1999, Science 284:770; Brennan and Brown, 2003,Breast Cancer Res. 5:69; Iso et al., 2003, Arterioscler. Thromb. Vasc.Biol. 23:543). Alternative intracellular pathways involving thecytoplasmic protein Deltex identified in Drosophila may also exist inmammals (Martinez et al., 2002, Curr. Opin. Genet. Dev. 12:524-33), andthis Deltex-dependent pathway may act to suppress expression of Wnttarget genes (Brennan et al., 1999, Curr. Biol. 9:707-710; Lawrence etal., 2001, Cur. Biol. 11:375-85).

Mammalian Notch receptors undergo cleavage to form the mature receptorand also following ligand binding to activate downstream signaling. Afurin-like protease cleaves the Notch receptors during maturation togenerate juxtamembrane heterodimers that comprise a non-covalentlyassociated extracellular subunit and a transmembrane subunit heldtogether in an auto-inhibitory state. Ligand binding relieves thisinhibition and induces cleavage of the Notch receptor by an ADAM-typemetalloprotease and a gamma-secretase, the latter of which releases theintracellular domain (ICD) into the cytoplasm, allowing it totranslocate into the nucleus to activate gene transcription. Cleavage byADAM occurs within the non-ligand binding cleavage domain within themembrane proximal negative regulatory region.

Hematopoietic stein cells (HSCs) are the best understood stem cells inthe body, and Notch signaling is implicated in their normal maintenanceas well as in leukemic transformation (Kopper & Hlajdu, 2004, PatholOncol. Res. 10:69-73). HSCs are a rare population of cells that residein a stromal niche within the adult bone marrow. These cells arecharacterized both by a unique gene expression profile as well as anability to continuously give rise to more differentiated progenitorcells to reconstitute the entire hematopoietic system. Constitutiveactivation of Notch1 signaling in HSCs and progenitor cells establishesimmortalized cell lines that generate both lymphoid and myeloid cells invitro and in long-term reconstitution assays (Varnum-Finney et al.,2000, Nat. Med. 6:1278-81), and the presence of Jagged1 increasesengraftment of human bone marrow cell populations enriched for HSCs(Karanu et al., 2000, J. Exp. Med. 192:1365-72). More recently, Notchsignaling has been demonstrated in HSCs in vivo and shown to be involvedin inhibiting HSC differentiation. Furthermore, Notch signaling appearsto be required for Wnt-mediated HSC self-renewal (Duncan et al., 2005.Nat. Immuol. 6:314).

The Notch signaling pathway also plays a central role in the maintenanceof neural stein cells and is implicated in their normal maintenance aswell as in brain cancers (Kopper & Hajdu, 2004, Pathol. Oncol. Res.10:69-73; Purow et al., 2005, Cancer Res. 65:2353-63; Hallahan et al.,2004, Cancer Res. 64:7794-800). Neural stein cells give rise to allneuronal and glial cells in the mammalian nervous system duringdevelopment, and more recently have been identified in the adult brain(Gage, 2000, Science 287:1433-8). Mice deficient for Notch1; the Notchtarget genes Hes1, 3, and 5; and a regulator of Notch signalingpresenilin1 (PS1) show decreased numbers of embryonic neural stem cells.Furthermore, adult neural stem cells are reduced in the brains of PS1heterozygote mice (Nakamura et al., 2000, J. Neurosci. 20:283-93;Hitoshi et al., 2002, Genes Dev. 16:846-58). The reduction in neuralstem cells appears to result from their premature differentiation intoneurons (Hatakeyama et al., 2004, Dev. 131:5539-50) suggesting thatNotch signaling regulates neural stem cell differentition andself-renewal.

Aberrant Notch signaling is implicated in a number of human cancers. TheNotch1 gene in humans was first identified in a subset of T-cell acutelymphoblastic leukemias as a translocated locus resulting in activationof the Notch pathway (Ellisen et al., 1991, Cell 66:649-61).Constitutive activation of Notch1 signaling in T-cells in mouse modelssimilarly generates T-cell lymphomas suggesting a causative role (Robeyet al., 1996, Cell 87:483-92; Pear et al., 1996, J. Exp. Med.183:2283-91; Yan et al., 2001, Blood 98:3793-9; Bellavia et al., 2000,EMBO J. 19:3337-48). Notch1 point mutations, insertions, and deletionsproducing aberrant Notch1 signaling have also been found to befrequently present in both childhood and adult T-cell acutelymphoblastic leukemia/lymphoma (Pear & Aster, 2004, Curr. Opin.Hemotol. 11:416-33).

The frequent insertion of the mouse mammary tumor virus into both theNotch1 and Notch4 locus in mammary tumors and the resulting activatedNotch protein fragments first implicated Notch signaling in breastcancer (Gallahan & Callahan, 1987, J. Virol. 61:66-74; Brennan & Brown,2003, Breast Cancer Res. 5:69; Politi et al., 2004, Semin. Cancer Biol.14:341-7). Further studies in transgenic mice have confirmed a role forNotch in ductal branching during normal mammary gland development, and aconstitutively active form of Notch4 in mammary epithelial cellsinhibits epithelial differentiation and results in tumorigenesis(Jhappan et al., 1992, Genes & Dev. 6:345-5; Gallahan et al., 1996,Cancer Res. 56:1775-85; Smith et al., 1995, Cell Growth Differ.6:563-77; Soriano et al., 2000, Int. J. Cancer 86:652-9; Uyttendaele etal., 1998, Dev. Biol. 196:204-17; Politi et al., 2004, Semin. CancerBiol. 14:341-7). Evidence for a role for Notch in human breast cancer isprovided by data showing the expression of Notch receptors in breastcarcinomas and their correlation with clinical outcome (Weijzen et al.,2002, Nat. Med. 8:979-86; Parr et al., 2004, Int. J. Mol. Med.14:779-86). Furthermore, overexpression of the Notch pathway has beenobserved in cervical cancers (Zagouras et al., 1995, PNAS 92:6414-8),renal cell carcinomas (Rae et al., 2000, Int. J. Cancer 88:726-32), headand neck squamous cell carcinomas (Leethanakul et al., 2000, Oncogene19:3220-4), endometrial cancers (Suzuki et al., 2000, Int. J. Oncol.17:1131-9), and neuroblastomas (van Limpt et al., 2000, Med. Pediatr.Oncol. 35:554-8), suggestive of a potential role for Notch in thedevelopment of a number of neoplasms. Interestingly, Notch signaling mayplay a role in the maintenance of the undifferentiated state ofApc-mutant neoplastic cells of the colon (van Es & Clevers, 2005, Trendsin Mol. Med. 11:496-502).

The Notch pathway is also involved in multiple aspects of vasculardevelopment including proliferation, migration, smooth muscledifferentiation, angiogenesis and arterial-venous differentiation (Isoet al., 2003, Arterioscler. Thromb. Vasc. Biol. 23:543). For example,homozygous null mutations in Notch1/4 and Jagged1 as well asheterozygous loss of DLL4 result in severe though variable defects inarterial development and yolk sac vascularization. Furthermore,DLL1-deficient and Notch2-hypomorphic mice embryos show hemorrhagingthat likely results from poor development of vascular structures (Galeet al., 2004, PVAS, 101:15949-54; Krebs et al., 2000, Genes Dev.14:1343-52; Xue et al., 1999, Hum. Mel. Genet. 8:723-30; Hrabe deAngelis et al., 1997, Nature 386:717-21; McCright et al., 2001, Dev.128:491-502). In humans, mutations in Jagged1 are associated withAlagille syndrome, a developmental disorder that includes vasculardefects, and mutations in Notch3 are responsible for an inheritedvascular dementia (Cadasil) in which vessel homeostasis is defective(Joutel et al., 1996, Nature 383:707-10).

Anti-Notch antibodies and their possible use as anti-cancer therapeuticshave been previously reported. See, e.g., U.S. Patent ApplicationPublication No. 2008/0131434, which is incorporated by reference hereinin its entirety. See also International Publication Nos. WO 2008/057144and WO 2008/076960, as well as U.S. Patent Application Publication Nos.2008/0226621, 2008/0118520, and 2008/0131908.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel Notch-binding agents and novelantagonists of one or more human Notch receptors, as well as methods ofusing those agents and antagonists. The present invention furtherprovides novel polypeptides, such as antibodies that bind one or morehuman Notch receptors, fragments of such antibodies, and otherpolypeptides related to such antibodies. In certain embodiments, theinvention provides antagonists of human Notch2 and/or human Notch3,including, but not limited to, antibodies that specifically bind humanNotch2 and/or human Notch3. As used herein, the phrase “Notch2 and/orNotch3” means “Notch2,” “Notch3,” or “both Notch2 and Notch3.” Incertain embodiments, the antibodies or other antagonists bind to aregion of the Notch receptor that is outside of the ligand-bindingdomain (e.g., EGF10 of Notch2 or EGF9 of Notch3). In certainembodiments, the antibodies specifically bind human Notch2. In certainembodiments, the antibodies specifically bind both human Notch2 andhuman Notch3. In some embodiments, the antibodies specifically bindhuman Notch3. Polynucleotides comprising nucleic acid sequences encodingthe polypeptides are also provided, as are vectors comprising thepolynucleotides. Cells comprising the polypeptides and/orpolynucleotides of the invention are further provided. Compositions(e.g., pharmaceutical compositions) comprising the novel Notchantagonists are also provided. Methods of using the agents andantagonists are also provided, such as methods of using the Notchantagonists to inhibit tumor growth, reduce the tumorigenicity oftumors, inhibit angiogenesis, and/or treat cancer or other diseasesassociated with angiogenesis.

In one aspect, the invention provides an agent (e.g., an antibody) thatspecifically binds to an EGF10 domain (or an equivalent of an EGF10domain) of one or more human Notch receptors. In certain embodiments,the agent is an antibody. In certain embodiments, the agent is anantagonist. In certain embodiments, the agent specifically binds toEGF10 of human Notch2 and/or EGF9 of human Notch3. EGF9 is the EGFwithin human Notch3 that is equivalent to EGF10 in the other human Notchreceptors Notch1, Notch 2, and Notch4. In some embodiments, the agentspecifically binds to EGF10 of Notch 2. In some embodiments, the agentspecifically binds to EGF10 of Notch 2 and to EGF9 of Notch 3. In someembodiments, the agent specifically binds to EGF9 of Notch 3. In otherembodiments, the agent binds to at least part of the sequence HKGAL (SEQID NO:28) within Notch2 EGF10. In some embodiments, the agent binds toat least part of the sequence HEDAI (SEQ ID NO:29) within Notch3 EGF9.

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the agent inhibits binding of a ligand to human Notch2and/or Notch3. In some embodiments, the agent inhibits binding of aligand to human Notch2. In some embodiments, the agent inhibits bindingof a ligand to Notch2 and Notch3. In other embodiments, the agentinhibits binding of a ligand to Notch3. In certain embodiments, theligand is DLL4, JAG1 or JAG2. In other embodiments, the agent inhibitssignaling of human Notch2 and/or Notch3. In some embodiments, the agentinhibits signaling of human Notch2. In some embodiments, the agentinhibits signaling of Notch2 and Notch3. In other embodiments, the agentinhibits signaling of Notch3. In some embodiments Notch2 and/or Notch3signaling is induced by DLL4, JAG1 or JAG2. Pharmaceutical compositionscomprising the agent and methods of using the agent for such uses asinhibiting angiogenesis, inhibiting tumor growth, reducing thetumorigenicity of a tumor, and/or treating cancer are also provided.

In a further aspect, the invention provides an antibody thatspecifically binds human Notch2 and/or Notch3, wherein the antibodycomprises (a) a heavy chain CDR1 comprising SSSGMS (SEQ ID NO:5), aheavy chain CDR2 comprising VIASSGSNTYYADSVKG (SEQ ID NO:6), and/or aheavy chain CDR3 comprising SIFYTT (SEQ ID NO:51); and/or (b) a lightchain CDR1 comprising RASQSVRSNYLA (SEQ ID NO:8), a light chain CDR2comprising GASSRAT (SEQ ID NO:9), and/or a light chain CDR3 comprisingQQYSNFPI (SEQ ID NO:10). In some embodiments, the antibody comprises (a)a heavy chain CDR comprising SSSGMS (SEQ ID NO:5), or a variant thereofcomprising 1, 2, 3, or 4 conservative amino acid substitutions; a heavychain CDR2 comprising VIASSGSNTYYADSVKG (SEQ ID NO:6), or a variantthereof comprising 1, 2, 3, or 4 conservative amino acid substitutions;and/or a heavy chain CDR3 comprising SIFYTT (SEQ ID NO:51), or a variantthereof comprising 1, 2, 3, or 4 conservative amino acid substitutions;and/or (b) a light chain CDR1 comprising RASQSVRSNYLA (SEQ ID NO:8), ora variant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; a light chain CDR2 comprising GASSRAT (SEQ ID NO:9), or avariant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; and/or a light chain CDR3 comprising QQYSNFPI (SEQ IDNO:10), or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions. Pharmaceutical compositions comprising the antibodyand methods of using the antibody for such uses as inhibitingangiogenesis, inhibiting tumor growth, reducing the tumorigenicity of atumor, and/or treating cancer are also provided.

In a further aspect, the invention provides an antibody thatspecifically binds human Notch2 and/or Notch3, wherein the antibodycomprises (a) a heavy chain CDR1 comprising SSSGMS (SEQ ID NO:5), aheavy chain CDR2 comprising VIASSGSNTYYADSVKG (SEQ ID NO:6), and/or aheavy chain CDR3 comprising GIFFAI (SEQ ID NO:7); and/or (b) a lightchain CDR1 comprising RASQSVRSNYLA (SEQ ID NO:8), a light chain CDR2comprising GASSRAT (SEQ ID NO:9), and/or a light chain CDR3 comprisingQQYSNFPI (SEQ ID NO:10). In certain embodiments, the antibodyspecifically binds Notch2. In some embodiments, the antibody comprises(a) a heavy chain CDR1 comprising SSSGMS (SEQ ID NO:5), or a variantthereof comprising 1, 2, 3, or 4 conservative amino acid substitutions;a heavy chain CDR2 comprising VIASSGSNTYYADSVKG (SEQ ID NO:6), or avariant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; and/or a heavy chain CDR3 comprising GIFFAI (SEQ IDNO:7), or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions; and/or (b) a light chain CDR1 comprisingRASQSVRSNYLA (SEQ ID NO:8), or a variant thereof comprising 1, 2, 3, or4 conservative amino acid substitutions; a light chain CDR2 comprisingGASSRAT (SEQ ID NO:9), or a variant thereof comprising 1, 2, 3, or 4conservative amino acid substitutions; and/or a light chain CDR3comprising QQYSNFPI (SEQ ID NO:10), or a variant thereof comprising 1,2, 3, or 4 conservative amino acid substitutions. Pharmaceuticalcompositions comprising the antibody and methods of using the antibodyfor such uses as inhibiting angiogenesis, inhibiting tumor growth,reducing the tumorigenicity of a tumor, and/or treating cancer are alsoprovided.

In another aspect, the invention provides an antibody that specificallybinds human Notch2 and/or Notch3, wherein the antibody comprises (a) aheavy chain CDR1 comprising SSSGMS (SEQ ID NO:5), a heavy chain CDR2comprising VIASSGSNTYYADSVKG (SEQ ID NO:6), and/or a heavy chain CDR3comprising (G/S)(I/S)F(F/Y)(A/P)(I/T/S/N) (SEQ ID NO:30); and/or (b) alight chain CDR1 comprising RASQSVRSNYLA (SEQ ID NO:8), a light chainCDR2 comprising GASSRAT (SEQ ID NO:9), and/or a light chain CDR3comprising QQYSNFPI (SEQ ID NO:10). In some embodiments, the antibodycomprises a heavy chain CDR3 comprising SIFYPT (SEQ ID NO:22). In someembodiments, the antibody comprises a heavy chain CDR3 comprisingSSSFFAS (SEQ ID NO:23). In other embodiments, the antibody comprises aheavy chain CDR3 comprising SSFYAS (SEQ ID NO:24). In certainembodiments, the antibody comprises a heavy chain CDR3 comprising SSFFAT(SEQ ID NO:25). In some embodiments, the antibody comprises a heavychain CDR3 comprising SIFYPS (SEQ ID NO:26). In yet other embodiments,the antibody comprises a heavy chain CDR3 comprising SSFFAN (SEQ IDNO:27). Pharmaceutical compositions comprising the antibody and methodsof using the antibody for such uses as inhibiting angiogenesis,inhibiting tumor growth, reducing the tumorigenicity of a tumor, and/ortreating cancer are also provided.

In another aspect, the invention provides a polypeptide that comprises:(a) a polypeptide (e.g., a heavy chain variable region) having at leastabout 80% sequence identity to SEQ ID NO:50, SEQ ID NO:14, SEQ ID NO:40,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56,SEQ ID NO:57, or SEQ ID NO:20 (with or without signal sequence); and/or(b) a polypeptide (e.g., a light chain variable region) having at leastabout 80% sequence identity to SEQ ID NO:13, SEQ ID NO:19 or SEQ IDNO:39 (with or without signal sequence). In certain embodiments, thepolypeptide is an antibody. In certain embodiments, the polypeptidespecifically binds human Notch2 and/or Notch3. In some embodiments, thepolypeptide specifically binds to human Notch2. In some embodiments, thepolypeptide binds to Notch2 and Notch3. In other embodiments, thepolypeptide binds to Notch3. In certain embodiments, the polypeptidecomprises a polypeptide having at least about 85%, at least about 90%,at least about 95%, at least about 98%, or about 100% sequence identityto SEQ ID NO:14, SEQ ID NO:13, or SEQ ID NO:50. Pharmaceuticalcompositions comprising the polypeptide and methods of using thepolypeptide for such uses as inhibiting angiogenesis, inhibiting tumorgrowth, reducing the tumorigenicity of a tumor, and/or treating cancerare also provided.

In still another aspect, the invention provides a polypeptide (e.g., anantibody or a heavy chain or light chain of an antibody) comprising: (a)a polypeptide having at least about 80%9 sequence identity to SEQ IDNO:49, SEQ ID NO:16, or SEQ ID NO:2 (with or without signal sequence);and/or (b) a polypeptide having at least about 80% sequence identity toSEQ ID NO:18, or SEQ ID NO:4 (with or without signal sequence. Incertain embodiments, the polypeptide comprises a polypeptide having atleast about 85%, at least about 90%, at least about 95%, at least about98%, or about 100% sequence identity to SEQ ID NO:39 or SEQ ID NO:40.Pharmaceutical compositions comprising the antibodies and methods oftreating cancer comprising administering therapeutically effectiveamounts of the antibodies are also provided.

In another aspect, the invention provides a polypeptide (e.g., anantibody or a heavy chain or light chain of an antibody) comprises: (a)a polypeptide having at least about 80% sequence identity to SEQ IDNO:50; and/or (b) a polypeptide having at least about 80% sequenceidentity to SEQ ID NO:13. In certain embodiments, the polypeptidecomprises a polypeptide having at least about 85%, at least about 90%,at least about 95%, at least about 98%, or about 100% sequence identityto SEQ ID NO:50 or SEQ ID NO:13. In certain embodiments, the polypeptideis an antibody that binds human Notch2 and/or human Notch3.Pharmaceutical compositions comprising the antibodies and methods oftreating cancer comprising administering therapeutically effectiveamounts of the antibodies are also provided.

In another aspect, the invention provides an antibody that comprises,consists, or consists essentially of a 59R1 IgG2 antibody comprising theheavy chain and light chain of SEQ ID NOs:16 and 18 (with or withoutsignal sequence), respectively, or as encoded by the DNA deposited withthe American Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va., USA, under the conditions of the Budapest Treaty on Oct.15, 2008, and assigned designation number PTA-9547. Pharmaceuticalcompositions comprising the antibody and methods of using the antibodyfor such uses as inhibiting angiogenesis, inhibiting tumor growth,reducing the tumorigenicity of a tumor, and/or treating cancer are alsoprovided.

In an additional aspect, the invention provides an antibody thatcomprises, consists or consists essentially of a 59R5 IgG2 antibodycomprising the heavy chain and light chain of SEQ ID NO:49 and SEQ IDNO:18 (with or without signal sequence), respectively, or as encoded bythe DNA deposited with the ATCC on Jul. 6, 2009, and assigneddesignation number PTA-10170. Pharmaceutical compositions comprising theantibody and methods of using the antibody for such uses as inhibitingangiogenesis, inhibiting tumor growth, reducing the tumorigenicity of atumor, and/or treating cancer are also provided.

In another aspect, the invention provides an antibody that competes forspecific binding to human Notch2 and/or Notch3 with an antibodycomprising a heavy chain variable region comprising SEQ ID NO:14 and alight chain variable region comprising SEQ ID NO:13. In certainembodiments, the antibody competes for specific binding with a 59R1 IgG2antibody comprising the heavy chain and light chain of SEQ ID NOs:16 and18 (with or without signal sequence), respectively, or as encoded by theDNA deposited with the ATCC on Oct. 15, 2008, and assigned designationnumber PTA-9547. In some embodiments, the antibody competes for bindingto human Notch2. In some embodiments, the antibody competes for bindingto human Notch2 and Notch3. In other embodiments, the antibody competesfor binding to human Notch3. Pharmaceutical compositions comprising theantibody and methods of using the antibody for such uses as inhibitingangiogenesis, inhibiting tumor growth, reducing the tumorigenicity of atumor, and/or treating cancer are also provided.

In a further aspect, the antibody competes for specific binding to humanNotch2 and/or Notch3 with an antibody comprising a heavy chain variableregion comprising SEQ ID NO:50 and a light chain variable regioncomprising SEQ ID NO:13. In some embodiments, the antibody competes forspecific binding with a 59R5 antibody comprising the heavy chain andlight chain of SEQ ID NOs: 49 and 18, respectively, or as encoded by theDNA deposited with the ATCC on Jul. 6, 2009, and assigned designationnumber PTA-10170. In some embodiments, the antibody competes for bindingto human Notch2. In some embodiments, the antibody competes for bindingto human Notch2 and Notch3. In other embodiments, the antibody competesfor binding to human Notch3. Pharmaceutical compositions comprising theantibody and methods of using the antibody for such uses as inhibitingangiogenesis, inhibiting tumor growth, reducing the tumorigenicity of atumor, and/or treating cancer are also provided.

In certain other aspects, the invention provides a polypeptide (with orwithout a signal sequence) comprising a sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:16, SEQ ID NO:18, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52 SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, as well as apolynucleotide encoding such a polypeptide. In certain embodiments, thepolypeptide is an antibody. In certain embodiments, the antibodyspecifically binds to human Notch2 and/or human Notch3. In certainembodiments, the antibody specifically binds to human Notch2. In certainembodiments, the antibody specifically binds to human Notch2 and humanNotch3. In certain embodiments, the antibody specifically binds to humanNotch3. In another aspect, the invention provides a polynucleotidecomprising a sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:47, SEQ ID NO:48, SEQID NO:58, SEQ ID NO: 59 and SEQ ID NO:60.

In another aspect, the invention provides a method of modulating thefunction of pericytes and/or vascular smooth muscle cells in a subject(e.g., at the site of a tumor or other aberrant angiogenesis in thesubject). In certain embodiments, the method comprises administering aneffective amount of an agent that specifically binds human Notch2 and/orhuman Notch3 to the subject. In certain embodiments, the agent is anantibody. In some embodiments, the agent is an antibody described in anyone of the aforementioned aspects and/or embodiments, as well as otheraspects and/or embodiments described herein. In certain embodiments, theagent is an antagonist. In certain embodiments, the agent specificallybinds to and is an antagonist of human Notch3. In certain embodiments,the modulation of the function of the pericytes and/or vascular smoothmuscle cells results in inhibition of angiogenesis and/or tumor growth.

In still another aspect, the invention provides a method of inhibitingangiogenesis (e.g., tumor angiogenesis) in a subject. In certainembodiments, the method comprises administering to the subject aneffective amount of an agent that specifically binds human Notch2 and/orhuman Notch3. In certain embodiments, the agent is an antagonist. Insome embodiments, the agent specifically binds to and is an antagonistof human Notch2. In certain embodiments, the agent specifically binds toand is an antagonist of human Notch3. In some embodiments, the agent isan antagonist of both Notch2 and Notch3. In some embodiments, theantagonist is an antibody. In certain embodiments, the agent is anantibody. In some embodiments, the agent is an antibody described in anyone of the aforementioned aspects and/or embodiments, as well as otheraspects and/or embodiments described herein. In some embodiments, theantagonist is not an antibody. In some embodiments, the method ofinhibiting angiogenesis further comprises administering to the subjectan antagonist of vascular endothelial cell growth factor (VEGF) or of aVEGF receptor. In certain embodiments, the method is a method ofinhibiting angiogenesis by modulating the function of pericytes and/orvascular smooth muscle cells.

In a further aspect, the invention provides a method of inhibitinggrowth of a tumor in a subject. In certain embodiments, the methodcomprises administering to the subject a therapeutically effectiveamount of an antagonist of human Notch2 and/or human Notch3. In certainembodiments, the antagonist is an antibody that specifically binds humanNotch2. In some embodiments, the antagonist is an antibody thatspecifically binds both human Notch2 and human Notch3. In certainembodiments, the antagonist is an antibody that specifically binds humanNotch3. In some embodiments, the antagonist is an antibody described inany one of the aforementioned aspects and/or embodiments, as well asother aspects and/or embodiments described herein. In certainembodiments, the tumor comprises a deletion or other mutation in thephosphatase and tensin homolog (PTEN) gene. In certain embodiments, thetumor is a breast tumor.

In a still further aspect, the invention provides a method of selectinga subject for treatment with a human Notch2 and/or human Notch3antagonist. In certain embodiments, the method comprises (a) determiningif the tumor comprises a deletion or mutation in the phosphatase andtensin homolog (PTEN) gene; and (b) selecting the subject for treatmentwith a Notch2 and/or Notch3 antagonist if the tumor comprises thedeletion or mutation. In some embodiments, the subject is treated with aNotch2 antagonist. In some embodiments, the subject is treated with anantagonist of Notch2 and Notch3. In some embodiments, the subject istreated with an antagonist of Notch3. In some embodiments the antagonistis an antibody. In certain embodiments, the tumor is a breast tumor.

In another aspect, the invention provides an antibody that specificallybinds to a non-ligand binding region of an extracellular domain of atleast one human Notch receptor (e.g., 1, 2, 3, or 4 Notch receptors). Incertain embodiments, the non-ligand binding region comprises or consistsof EGF repeat 10 of a human Notch receptor (or an equivalent of EGF10,such as EGF9 of human Notch3). In some embodiments, the antibodyinhibits tumor growth. In some embodiments, the antibody inhibitsbinding of a ligand to a Notch receptor. In certain embodiments, theantibody inhibits signaling by the Notch receptor. In some embodiments,the Notch receptor is a human Notch1, Notch2, Notch3, or Notch4receptor. In certain embodiments, the antibody specifically binds toNotch2 (for example, EGF10 of Notch2). In certain embodiments, theantibody specifically binds to Notch2 and at least one additional Notchreceptor. In certain embodiments, the additional Notch receptor isNotch3. Pharmaceutical compositions comprising the antibodies andmethods of treating cancer comprising administering therapeuticallyeffective amounts of the antibodies are also provided.

In an additional aspect, the invention provides an antibody thatspecifically binds to two or more (i.e., at least two or two, three, orfour) human Notch receptors. In certain embodiments, the antibodyspecifically binds to a non-ligand binding region of an extracellulardomain of the two or more human Notch receptors. In certain embodiments,if the two or more human Notch receptors comprise Notch1, Notch2, orNotch4, the antibody binds to EGF10 of Notch1, Notch2, or Notch4, and ifthe two or more human Notch receptors comprise Notch3, the antibodybinds to EGF9 of Notch3. In certain embodiments, the non-ligand bindingregion is not EGF4. In certain embodiments, the two or more human Notchreceptors comprise Notch2. In certain embodiments, the two or more humanNotch receptors comprise Notch3. In still further embodiments, the twoor more human Notch receptors comprise Notch2 and Notch3. In certainembodiments, the antibody is an antagonist of the two or more humanNotch receptors. In certain embodiments, the antibody inhibits tumorgrowth. Pharmaceutical compositions comprising the antibodies andmethods of treating cancer comprising administering therapeuticallyeffective amounts of the antibodies are also provided.

In yet another aspect, the invention provides an isolated antibody thatspecifically binds to a non-ligand binding region of an extracellulardomain of a human Notch2 receptor and inhibits tumor growth, wherein thenon-ligand binding region comprises or consists of EGF repeat 10 of thehuman Notch2 receptor (e.g., SEQ ID NO:36). In some embodiments, theantibody does not bind to any region of human Notch2 outside of EGFrepeat 10. In certain embodiments, the antibody also specifically bindsto EGF repeat 10 (or equivalent) of at least one additional human Notchreceptor (e.g., EGF9 of Notch3). In some embodiments, the antibody bindsto human Notch2 EGF10 and Notch3 EGF9. Pharmaceutical compositionscomprising the antibodies and methods of treating cancer comprisingadministering therapeutically effective amounts of the antibodies arealso provided.

In yet another aspect, the invention provides an isolated antibody thatspecifically binds to a non-ligand binding region of an extracellulardomain of a human Notch3 receptor and inhibits tumor growth, wherein thenon-ligand binding region comprises or consists of EGF repeat 9 of thehuman Notch3 receptor (equivalent to EGF10 in the other Notchreceptors). In some embodiments, the antibody does not bind to anyregion of human Notch3 outside of EGF repeat 9. In certain embodiments,the antibody also specifically binds to EGF repeat 10 of at least oneadditional human Notch receptor. In some embodiments, the antibody bindsto human Notch3 EGF9 and Notch2 EGF10. Pharmaceutical compositionscomprising the antibodies and methods of treating cancer comprisingadministering therapeutically effective amounts of the antibodies arealso provided.

In a still further aspect, the invention provides an antibody that bindsa non-ligand binding region of an extracellular domain of a human Notchreceptor and comprises: (a) a heavy chain CDR1 comprising SSSGMS (SEQ IDNO:5), a heavy chain CDR2 comprising VIASSGSNTYYADSVKG (SEQ ID NO:6),and/or a heavy chain CDR3 comprising GIFFAI (SEQ ID NO:7); and/or (b) alight chain CDR1 comprising RASQSVRSNYLA (SEQ ID NO:8), a light chainCDR2 comprising GASSRAT (SEQ ID NO:9), and/or a light chain CDR3comprising QQYSNFPI (SEQ ID NO:10). In certain embodiments, the humanNotch receptor is Notch2. In certain embodiments, the antibody binds toEGF10 of a human Notch2 receptor and/or EGF9 of a human Notch3 receptor.In an additional aspect, the invention provides an antibody thatcompetes with such an antibody for specific binding to a non-ligandbinding region of an extracellular domain of Notch2 in a competitivebinding assay. Pharmaceutical compositions comprising the antibodies andmethods of treating cancer comprising administering therapeuticallyeffective amounts of the antibodies are also provided. Methods ofinhibiting angiogenesis comprising administering the compositions arealso provided.

In another aspect, the invention provides an antibody that binds anon-ligand binding region of an extracellular domain of a human Notchreceptor and comprises: (a) a heavy chain CDR1 comprising SSSGMS (SEQ IDNO:5), a heavy chain CDR2 comprising VIASSGSNTYYADSVKG (SEQ ID NO:6),and/or a heavy chain CDR3 comprising SIFYTT (SEQ ID NO:51); and/or (b) alight chain CDR1 comprising RASQSVRSNYLA (SEQ ID NO:8), a light chainCDR2 comprising GASSRAT (SEQ ID NO:9), and/or a light chain CDR3comprising QQYSNFPI (SEQ ID NO:10). In certain embodiments, the humanNotch receptor is Notch2. In some embodiments, the antibody binds to thehuman Notch2 and Notch3 receptors. In certain embodiments, the antibodybinds to EGF10 of a human Notch2 receptor and/or EGF9 of a human Notch3receptor. In another embodiment, the invention provides an antibody thatcompetes with such an antibody for specific binding to a non-ligandbinding region of an extracellular domain of Notch2 in a competitivebinding assay. Pharmaceutical compositions comprising the antibodies andmethods of treating cancer comprising administering therapeuticallyeffective amounts of the antibodies are also provided. Methods ofinhibiting angiogenesis comprising administering the compositions arealso provided.

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the antibody specifically binds to both human Notch2and human Notch3.

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the antibody is a recombinant antibody. In certainembodiments, the antibody is a monoclonal antibody. In certainembodiments, the antibody is a chimeric antibody. In certainembodiments, the antibody is a humanized antibody. In certainembodiments, the antibody is a human antibody. In some embodiments, theantibody is monovalent, bivalent or multivalent. In certain embodiments,the antibody is a monospecific antibody. In certain embodiments, anindividual antigen-binding site of the antibody binds (or is capable ofbinding) a non-ligand binding region of the extracellular domain of morethan one human Notch receptor (e.g., Notch2 and Notch3). In certainalternative embodiments, the antibody is a bispecific antibody. Incertain embodiments, the antibody is an IgG1 antibody. In certainembodiments, the antibody is an IgG2 antibody. In certain embodiments,the antibody is conjugated to a cytotoxic moiety. In certainembodiments, the antibody is isolated. In still further embodiments, theantibody is substantially pure.

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the cancer or tumor treated with the antibody is abreast, colorectal, lung, pancreatic, prostate, or head and neck canceror tumor. In certain embodiments, the cancer or tumor is melanoma. Incertain embodiments, the cancer or tumor is a breast cancer or tumor. Incertain embodiments, the cancer or tumor is a colorectal cancer ortumor. In certain embodiments, the cancer or tumor is a pancreaticcancer or tumor. In certain embodiments, the cancer or tumor is aprostate cancer or tumor.

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the methods of treating cancer comprise inhibitingtumor growth. In certain embodiments, the methods of treating cancercomprise reducing the tumorigenicity of tumors (e.g., by reducing thefrequency of cancer stem cells in the tumor).

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the antagonist or antibody is administered to asubject in combination with an additional treatment for cancer. Incertain embodiments, the additional treatment for cancer comprisesradiation therapy, chemotherapy, and/or an additional antibodytherapeutic. In some embodiments, the additional treatment for cancercomprises a chemotherapeutic agent. In certain embodiments, thechemotherapy comprises paclitaxel (e.g., TAXOL), irinotecan,gemcitabine, and/or oxaliplatin. In certain embodiments, the additionalantibody therapeutic is an antibody that specifically binds a humanNotch receptor (e.g., Notch1, 2, 3, or 4) or a human Notch receptorligand (e.g., DLL4 or JAG1). In some embodiments, the additionalantibody therapeutic is an anti-DLL4 antibody. In certain alternativeembodiments, the additional antibody therapeutic is an antibody thatspecifically binds vascular endothelial cell growth factor (VEGF). Incertain embodiments, the additional therapeutic binds a VEGF receptor.

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the antibody is administered to a subject incombination with a second therapeutic agent that is an anti-angiogenicagent.

Cell lines (e.g., hybridoma cell lines) comprising or producing theantibodies or other polypeptides described herein are further providedby the invention. Polynucleotides (e.g., vectors) comprising thepolynucleotides described herein, including polynucleotides encoding thepolypeptides or the light chain variable regions or heavy chain variableregions of the antibodies described herein are also provided, as arecell lines comprising such polynucleotides.

In certain embodiments, the present invention provides a method oftreating cancer, wherein the cancer comprises cancer stem cells,comprising administering to the subject a therapeutically effectiveamount of an antibody which binds a Notch receptor. In a more particularaspect, the present invention provides a method of treating cancer,wherein the cancer comprises stem cells expressing one or more Notchreceptor family members, comprising administering to the subject atherapeutically effective amount of an antibody that binds those Notchreceptor family members. The present invention provides antibodies thatbind to the non-ligand binding domain of the extracellular domain of ahuman Notch receptor and are therapeutically effective against cancer.Thus, in certain embodiments, the present invention provides an antibodythat specifically binds to a non-ligand binding region of theextracellular domain of a human Notch receptor and that inhibits tumorgrowth. In certain embodiments, the present invention further provides amethod of treating cancer, the method comprising administering atherapeutically effective amount of an antibody that specifically bindsto a non-ligand binding region of the extracellular domain of a humanNotch receptor protein and inhibits tumor growth.

Various advantages in using an antibody that binds Notch receptor familymembers or the ligands to those Notch receptors to treat such cancersare contemplated herein. In some embodiments, certain Notch receptorsare highly expressed in certain solid tumors, for example, breast andcolon, and this provides a sink for active drug where the drug binds tothe Notch receptor. Antibodies that bind overexpressed Notch receptorsare anticipated to have a better safety profile than currently availablechemotherapeutic drugs.

The invention further provides a method of treating cancer in a human,wherein the cancer comprising cancer stem cells is not characterized byoverexpression by the cancer stem cell of one or more Notch receptors,comprising administering to the human a therapeutically effective amountof an antibody which binds to a Notch receptor and blocks ligandactivation of a Notch receptor.

The invention further provides a method of treating cancer in a humancomprising administering to the human therapeutically effective amountsof (a) a first antibody which binds a Notch receptor and inhibits growthor proliferation of cancer stem cells which overexpress Notch receptors;and (b) a second antibody which binds a Notch receptor and blocks ligandactivation of a Notch receptor.

The invention also provides a method of treating cancer, wherein thecancer is selected from the group consisting of breast, colon, rectaland colorectal cancer, comprising administering a therapeuticallyeffective amount of an antibody which binds Notch. The invention alsoprovides another method of treating cancer, wherein the cancer isselected from the group consisting of breast, colon, pancreatic,prostate, lung, rectal and colorectal cancer, comprising administering atherapeutically effective amount of an antibody that blocks ligandactivation of a Notch receptor. The invention also provides stillanother method of treating cancer, wherein the cancer is selected fromthe group consisting of breast, colon, pancreatic, prostate, lung,rectal and colorectal cancer, comprising administering a therapeuticallyeffective amount of an antibody that binds Notch and an antibody thatblocks ligand activation of a Notch receptor.

In further embodiments, the invention provides articles of manufacturefor use (among other things) in the above methods. For example, theinvention provides an article of manufacture comprising a container anda composition contained therein, wherein the composition comprises anantibody that binds Notch, and further comprises a package insertindicating that the composition can be used to treat a cancer comprisingcancer stem cells. In some embodiments, the invention provides anarticle of manufacture comprising a container and a compositioncontained therein, wherein the composition comprises an antibody thatbinds Notch, and further comprises a package insert indicating that thecomposition can be used to treat cancer comprising cancer stem cellsthat express one or more Notch receptors.

In certain embodiments, the invention additionally pertains to anarticle of manufacture comprising a container and a compositioncontained therein, wherein the composition comprises an antibody whichbinds a Notch receptor and blocks ligand activation of a Notch receptor,and further comprises a package insert indicating that the compositioncan be used to treat cancer, wherein the cancer comprises cancer stemcells that are not characterized by overexpression of the Notchreceptor.

In certain embodiments, an article of manufacture is provided whichcomprises (a) a first container with a composition contained therein,wherein the composition comprises a first antibody that binds a Notchreceptor and inhibits growth of cancer cells comprising cancer stemcells overexpressing Notch; and (b) a second container with acomposition contained therein, wherein the composition comprises asecond antibody which binds Notch and blocks ligand activation of aNotch receptor.

In some embodiments, a further article of manufacture is provided whichcomprises a container and a composition contained therein, wherein thecomposition comprises an antibody which binds Notch and blocks ligandactivation of a Notch receptor, and further comprises a package insertindicating that the composition can be used to treat a cancer selectedfrom the group consisting of colon, pancreatic, prostate, lung, rectaland colorectal cancer.

The invention additionally provides: an antibody (e.g., a human antibodyor a humanized antibody) which binds Notch and blocks ligand activationof a Notch receptor; a composition comprising the antibody and apharmaceutically acceptable carrier; and an immunoconjugate comprisingthe antibody conjugated with a cytotoxic agent.

In one aspect, the invention provides an isolated polynucleotideencoding any of the antibodies or polypeptides of the aforementionedaspects or embodiments, as well as other aspects and/or embodimentsdescribed elsewhere herein. In some embodiments, the invention providesa vector comprising the polynucleotide. In some embodiments, a host cellcomprises the polynucleotide or the vector. In other embodiments, aprocess of producing the antibody comprises culturing a host cellcomprising the polynucleotide so that the polynucleotide is expressedand, optionally, further comprises recovering the antibody from the hostcell culture (e.g., from the host cell culture medium).

Moreover, the invention provides an isolated polynucleotide encoding ahumanized or human antibody as described in the aforementionedembodiments or aspects, as well as described elsewhere herein; a vectorcomprising the polynucleotide; a host cell comprising the polynucleotideor the vector; as well as a process of producing the antibody comprisingculturing a host cell comprising the polynucleotide so that thepolynucleotide is expressed and, optionally, further comprisingrecovering the antibody from the host cell culture (e.g., from the hostcell culture medium).

The invention further pertains to an immunoconjugate comprising anantibody that binds Notch conjugated to one or more calicheamicinmolecules, and the use of such conjugates for treating a Notchexpressing cancer, e.g., a cancer in which cancer stern cellsoverexpress Notch.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, including, but notlimited to, groups of alternatives separated by “and/or” or “or,” thepresent invention encompasses not only the entire group listed as awhole, but each member of the group individually and all possiblesubgroups of the main group, but also the main group absent one or moreof the group members. The present invention also envisages the explicitexclusion of one or more of any of the group members in the claimedinvention. For example, language such as “X and/or Y” encompasses “X”individually, “Y” individually, as well as “X” and “Y” together.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: 59R1 antibodies and variants bind human Notch2 and block ligandbinding. (A) FACS analysis of binding by 59R1 Fab to human Notch2.“Clone 1” is 59R1 Fab which was shown to bind human Notch2 on stablytransfected HEK293 cells. “Clone 5” is the Fab of a different cloneisolated from the phage library which did not bind Notch2. (B) FACSanalysis of blocking of ligand (JAG1) binding by 59R1 Fab. “Clone 1” is59R1 Fab which was shown to block binding of a hJagged1 ECD-Fc fusion tohuman Notch2 on stably transfected HEK293 cells. “Clone 5” is the Fab ofa different clone isolated from the phage library which did not blockligand binding in the assay. (C) FACS analysis of binding of 59R1 IgG2antibody to human Notch2 on stably transfected HEK293 cells. 59R1 IgG2antibody was shown to bind human Notch2 on stably transfected HEK293cells. (D) FACS analysis of blocking of ligand (DLL4) binding by 59R1IgG2 antibody. 59R1 IgG2 antibody was shown to block binding of a hDLL4ECD-Fc fusion to human Notch2 on stably transfected HEK293 cells. (E)Affinity maturation strategy for heavy chain CDR3 of 59R1. The parentalsequence of the heavy chain CDR3 of 59R1 is shown boxed. Allowed residuechanges are as indicated below the parental sequence in the figure. (F)Screening of affinity maturated 59R1 sequences for JAG1 blockingability. Improved variants are indicated with arrows.

FIG. 2: FACS analysis of cross-reactivity of the 59R1 IgG² antibody tothe four human Notch homologues. 59R1 was found to bind hNotch2 andhNotch3 on transiently transfected HEK-293 cells but was found to notexhibit significant binding to hNotch1 and hNotch4 on the same cells.

FIG. 3: Epitope mapping of 59R1 antibody. (A) Anti-Notch2/3 antibody59R1 binds to EGF repeat 10 of human Notch2. Supernatant from HEK 293cells expressing recombinant Notch2-Fc fusion proteins with theindicated EGF repeats of Notch2 between 1 and 12 (x-axis) were used inELISA with anti-Notch2/3 antibody 59R1. The OD (y-axis) indicatedantibody binding (hatched bars) only to Notch2 fusion proteinscomprising EGF repeat 10. (The figure shows data obtained from twoseparate experiments which are shown separately in the top and bottomgraphs.) (B) EGF Repeats 11 and 12 are not involved in anti-Notch2/3antibody 59R1 binding to fill length hNotch2. FACS analysis of HEK 293cells transfected with green fluorescent protein (GFP) (x-axis) alone(top left) or co-transfected with GFP and either full length Notch2intact or with full length Notch 2 with EGF repeat 11 deleted (ΔEGF11)or EGF repeat 12 deleted (ΔEGF12). Binding of 59R1 is indicated alongthe y-axis (PE) to all three Notch2 proteins in GFP-expressing cells.(C) EGF repeat 10 is involved in anti-Notch2/3 antibody 59R1 binding tofull-length hNotch2, but not in ligand binding. Binding by ananti-Notch2 antibody 59M70 that binds to EGF1-4 of hNotch2 is indicatedas “anti-Notch2 binding.” Binding by DLL4 is indicated as “ligandbinding.”

FIG. 4: Anti-Notch2/3 antibody 59R1 inhibits Notch2 signaling inluciferase reporter assays. (A) 59R11 blocks hDLL4-induced Notch2reporter activity. (B) 59R1 blocks hJAG1-induced Notch2 reporteractivity (C) 59R1 blocks hJAG2-induced Notch2 reporter activity

FIG. 5: Notch2/3 Receptor Antibody 59R1 Inhibits Tumor Formation andGrowth in vivo. (A) Anti-Notch2/3 (59R1) Inhibits the Formation of PE13Breast Tumors. NOD/SCID mice injected with PE13 breast tumor cells weretreated with control antibody (squares) or anti-Notch2/3 antibody 59R1(open triangles) two days after cell injection and tumor volume (y-axis,mm³) was measured across time (x-axis, days post cell injection).Treatment with 59R1 antibodies significantly inhibited tumor formationcompared to control. (p<0.001) (B) Anti-Notch2/3 (59R1) InhibitsFormation of T3 Breast Tumors. NOD/SCID mice injected with T3 breasttumor cells were treated with control antibody (squares) oranti-Notch2/3 antibody 59R1 (open triangles) two days after cellinjection, and tumor volume (y-axis, mm³) was measured across time(x-axis, days post cell injection). Treatment with 59R1 antibodiessignificantly inhibited tumor formation compared to control. (p<0.001)(C) Anti-Notch2/3 (59R1) Inhibits the Growth of Colo-205 Colon Tumors.6-8 week-old immunodeficient bg/nu XID female mice on a Swiss CD-1background injected with Colo-205 colon tumor cells were treated withcontrol antibody (squares) or anti-Notch2/3 antibody 59R (diamonds)after tumor volume reached a size between 65 to 200 mm³. Mean tumorvolume (y-axis, mm³) was measured across time (x-axis, days post cellinjection). Treatment with 59R1 antibodies inhibited tumor growthcompared to control (*** p<0.001 after day 40). (D) Anti-Notch2/3 (59R1)Inhibits the Growth of PN4 Pancreatic Tumors. NOD/SCID mice injectedwith PN4 pancreatic tumor cells were treated with control antibody(squares) or anti-Notch2/3 antibody 59R1 (diamonds) after tumor volumereached an a size between 65 to 200 mm³. Mean tumor volume (y-axis, mm³)was measured across time (x-axis, days post cell injection). Treatmentwith 59R1 antibodies inhibited tumor growth compared to control (***p<0.001 after day 70). (E) Anti-Notch2/3 (59R1) Inhibits the Growth ofPE13 Breast Tumors. NOD/SCID mice injected with PE13 breast tumor cellswere treated with control antibody (squares) or anti-Notch2/3 antibody59R1 (diamonds) after tumor volume reached a size between 65 to 200 mm³.Mean tumor volume (y-axis, mm³) was measured across time (x-axis, dayspost cell injection). Treatment with 59R1 antibodies inhibited tumorgrowth compared to control (* p<0.05 after day 57). (F) Anti-Notch2/3(59R1) Inhibits the Growth of T3 Breast Tumors. NOD/SCID mice injectedwith T3 breast tumor cells were treated with control antibody (solidbars) or anti-Notch2/3 antibody 59R1 (open bars) after tumor volumereached a size between 65 to 200 mm³. Mean tumor volume was measured ondays 18, 25, 39, and 42 post cell injection. Treatment with 59R1antibodies inhibited tumor growth compared to control (*** p<0.001 onday 42).

FIG. 6: Anti-Notch2/3 antibody 59R1 delays B51 breast tumor recurrenceafter paclitaxel treatment.

FIG. 7: Anti-Notch2/3 antibody 59R1 decreases cancer stem cell frequencyin B51 breast tumor.

FIG. 8: In combination with gemcitabine, anti-Notch2/3 antibody 59R1inhibits the growth of PN4 pancreatic tumors.

FIG. 9: Anti-Notch2/3 antibody 59R1 inhibits tumor growth in an M4melanoma xenograft model.

FIG. 10: Anti-Notch2/3 antibody 59R1 inhibits the growth of C28 colontumors alone and in combination with irinotecan.

FIG. 11: 59R1 IgG2 antibody significantly inhibits tumor growth ofestablished human tumor xenografts in vivo. Established Colo-205 (A), C8(B), PN8 (C), B34 (D), B39 (E), B44 (F), PE-13 (G) and T1 (H) tumors(s.c, n=10 per group) were treated at 15 mg/kg once a week with theindicated antibodies (1B711, LZ-1 control antibody, black squares; 59R1,black triangles; AVASTIN, black circles; AVASTIN+59R1, black diamonds).Tumor volume (x-axis) is plotted over time (y-axis). In the Colo-205xenograft model, combination therapy of 59R1 with AVASTIN wassignificantly more effective than either antibody treatment alone. InFIGS. 11B-11H, asterisks indicate significant tumor-growth inhibition atday shown: *, P<0.05; **, P<0.01; ***, P<0.001, Student's t-test;Symbols, mean; bars, SEM.

FIG. 12: Relative expression levels of selected genes are significantlyregulated by 59R1 treatment in various xenograft tumor models.Expression levels of HEYL (A), Notch3 (B), RGS5 (C), ANGPT1 (D) andANGPT2 (E) were individually tested by TaqMan analysis from previouslytested xenograft models. Notably, lack of estrogen (ne) abrogates effectof 59R1 in reducing ANGPT1 and ANGPT2 expression in host stroma of T1harboring mice. Open circles correspond to individual tumors analyzed.Horizontal line, mean.

FIG. 13: The tumor suppressor PTEN gene is deleted in many of the breasttumors in which 59R1 showed anti-tumor efficacy. The PTEN exon,Affymetrix probe distribution, and the deletions in the PTEN gene inchromosome 10 are shown. The thick and thin gray-shaded bars indicatethe homozygous and heterozygous deletions of the chromosome fragments,respectively.

FIG. 14. Epitope mapping of 59R1 antibody. (A) Protein alignment ofhuman Notch homologues. The alignment was performed by Clone ManagerSoftware. The EGF 10 repeat of human Notch1 (residues 356-418 of SEQ IDNO:45), Notch2 (residues 359-421 of SEQ ID NO:31), and Notch4 (residues376-439 of SEQ ID NO:46) and the equivalent EGF in human Notch3, EGF9,(residues 335-397 of SEQ ID NO:32), is as indicated. The boxed areaindicates a region containing one or more amino acid(s) that make up atleast part of the 59R1 epitope as defined by FACS binding of 59R1 IgG2antibody to an hNotch2 H385N AL 388-89 SN mutant (FIG. 14B) and to anhNotch1 construct in which aa 382-386 have been mutated to correspond tothe hNotch2 sequence (FIG. 14C). (B) 59R1 IgG2 antibody binds tohNotch2, but not a mutant hNotch2 in which certain EGF 10 residues havebeen mutated to hNotch1 residues (H385N AL 388-89 SN). (C) 59R1 IgG2antibody does not bind to hNotch1, but does bind to a mutant hNotch1 inwhich certain EGF 10 residues (aa 382-387) have been mutated to matchthe hNotch2 residues 385-389.

FIG. 15. In vitro characterization of 59R5. (A) FIG. 15A shows thatantibody 59R5 is able to block ligand-induced signaling of Notch2 andNotch3. PC3 tumor cells were transiently transfected with human or mouseNotch receptor (hN2, human Notch2; mN2, murine Notch2; hN3, humanNotch3; mN3, murine Notch3) and GFP inducible reporter construct.Transfected cells were incubated with different concentrations ofantibody 59R1 and 59R5 in the presence of passively immobilized DLL4 Fc.(B) FIG. 15B shows that 59R5 binds to a similar epitope as 59R1. HEK 293cells were transiently transfected with expression vectors encodinghuman Notch2, human Notch1, or human Notch1 with residues 382-386mutated to the corresponding human Notch2 residues. Cells were alsoco-transfected with a plasmid encoding green fluorescent protein (GFP)to mark those cells that received transfected plasmid. Cells wereincubated with 59R1 or 59R5 and fluorescent secondary antibody and thenexamined by FACS. The regions highlighted by the boxes suggest thatcells transfected with the indicated Notch expression vector were ableto bind to 59R1 or 59R5.

FIG. 16. Notch receptor antibody 59R5 inhibits tumor formation andgrowth in vivo. FIG. 16A shows in vivo treatment of PE13 breast tumorcells with antibody 59R5. FIG. 16B shows in vivo treatment of C28 coloncells with antibody 59R5. FIG. 16C shows in vivo treatment of Colo205colon cells with antibody 59R5.

FIG. 17. In vivo treatment of tumors using Notch2/3 antibody 59R5 incombination treatment. (A) Mice were injected with PN8 pancreatic tumorcells. Tumors were allowed to grow for 33 days until they had reached anaverage volume of 120 mm3. The animals were treated with gemcitabine at20 mg/kg once per week for four week in combination with either controlAb (squares), 59R1 (triangles), or 59R5 (circles). (B) Mice wereinjected with PE13 breast tumor cells. Tumors were allowed to grow for40 days before treatments were initiated. The animals were treated withTAXOL at 15 mg/kg twice per week for 5 weeks, plus either controlantibody (squares) or 59R5 (circles). After 5 weeks, the TAXOLtreatments were stopped and the antibody treatments continued.

FIG. 18. Regulation of gene expression in tumors after treatment withantibody 59R5. FIG. 18 shows expression levels of selected genes instromal cells and selected human genes in PE13 tumor cells aftertreatment with 59R1, 59R5, or control antibody.

FIG. 19. Reduction of PE13 breast cancer stem cell frequency by 59R1.(A) Established tumors were treated with control antibody, taxol pluscontrol antibody, 59R1, or taxol plus 59R1. Tumors were harvested afterthree weeks of treatment, processed and serial titrations of human cellsfrom each the four treatment groups were transplanted into a new set ofmice (n=10 per cell dose). Tumor growth rate was determined after 75days. Tumor growth rate after 75 days of growth was used to calculatethe CSC frequency using the L-calc program (Stem Cell Technologies,Inc.). (B) Cancer stem cell frequency in PE13 breast tumors aftertreatment with 59R1 and/or taxol. (C) Cancer stem cell frequency in PN4pancreatic tumors after treatment with 59R1 and/or gemcitabine. (D)Cancer stem cell frequency in PE13 breast tumors after treatment with59R5 and/or taxol. A single asterisk indicates a statisticallysignificant difference (p<0.05) vs. the control antibody treated groupand a double asterisk indicates a significant difference vs. the taxoland control antibody treated group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel agents, including, but not limitedto polypeptides such as antibodies, that bind to one or more human Notchreceptors, such as Notch2 and/or Notch3. The Notch-binding agentsinclude antagonists of the human Notch receptor(s). Related polypeptidesand polynucleotides, compositions comprising the Notch-binding agents,and methods of making the Notch-binding agents are also provided.Methods of using the novel Notch-binding agents, such as methods ofinhibiting tumor growth, inhibiting angiogenesis, and/or treating canceror other angiogenesis-related disease, are further provided.

The present invention further identifies molecules (e.g., antibodies)that specifically bind to a non-ligand binding region of theextracellular domain of a human Notch receptor and inhibit tumor growthin vivo. The ligand binding region of Notch, which is necessary andsufficient for ligand binding, has been identified as EGF repeats 11 and12, suggesting this region of the Notch receptor is important in Notchsignaling and tumorigenesis (Rebay et al., 1991, Cell 67:687; Lei etal., 2003, Dev. 130:6411; Hambleton et al., 2004, Structure 12:2173).Unexpectedly, antibodies that bind outside the ligand binding domain ofthe extracellular domain of human Notch receptor have been found toinhibit tumor cell growth in vivo (see U.S. Patent Publication No.2008/0131434, incorporated by reference herein in its entirety). Thus,antibodies that bind outside the ligand binding domain of theextracellular domain of one or more of the human Notch receptors—Notch1,Notch2, Notch3, and Notch4—have value as potential cancer therapeutics.

An antibody that specifically binds to an epitope containing residueswithin EGF repeat 10 of human Notch2 has now been identified (Examples 1and 3 and FIGS. 3A-3C). The antibody, 59R1, inhibits binding of ligandto Notch2 (Example 1 and FIGS. 1A-1D) and inhibits ligand-induced Notch2signaling (Example 4 and FIG. 4A-4C), despite binding to Notch2 in aregion outside of the ligand-binding region. 59R1 also specificallybinds human Notch3 (Example 2 and FIG. 2). The antibody has been foundto prevent or inhibit tumor cell growth in vivo in a variety ofdifferent xenograft models, either alone or in combination with a secondanti-cancer agent (Examples 5, 6, 7, and 9 and FIGS. 5A-F, 6, 8-10, and11A-H). The antibody has also been shown to reduce the tumorigenicity ofa tumor in vivo in multiple xenograft models by reducing the frequencyof cancer stem cells (Examples 8 and 23 and FIGS. 7 and 19A-C). Inaddition, treatment with 59R1 was found to downregulate expression ofRGS5 (a marker for pericytes and/or vascular smooth muscle cells),Notch3, and HeyL in the stroma of various tumors (Example 10 and FIGS.12A-E) and to upregulate hypoxia in breast and colon tumors (Example11). Without being bound by theory, these data indicate that the 59R1antibody has an inhibitory effect on tumor angiogenesis that is due, atleast in part, to modulation of the function of pericytes and/orvascular smooth muscle cells. Treatment with 59R1 was also found toregulate additional genes in breast tumors. Cell cycle gene pathways,myc-activating genes and several stem cell gene sets were found to bedown-regulated by 59R1 (Example 22).

An additional human antibody, 59R5, has also been developed. 59R5 hasproperties that are similar to 59R1, such as similar binding affinity toNotch2 and Notch3 and similarities or overlap in their epitopes (Example13 and FIG. 15B). Antibody 59R5 has been shown to have similar activityas 59R1 in blocking Notch2 and Notch 3 signaling (Example 13 and FIG.15A). The 59R5 antibody has also been shown to inhibit tumor growth invivo in several xenograft models, either alone or in combination with asecond anti-cancer agent (Examples 14 and 15 and FIGS. 16A-C and 17A-B).In addition, treatment with 59R5, like 59R1, was found to downregulateexpression of RGS5, Notch3, and HeyL in the stroma of various tumors,and 59R5 was also found to regulate the expression of human genes ID4,EDNRA, and EGLN3 in tumor cells to a similar extent as 59R1 (Example16). 59R5 was further shown to reduce the tumorigenicity in vivo in axenograft model by reducing the frequency of cancer stem cells (Example23 and 19D).

DEFINITIONS

An “antagonist” of a Notch receptor is a term that includes any moleculethat partially or fully blocks, inhibits, or neutralizes a biologicalactivity of the Notch pathway. Suitable antagonist moleculesspecifically include antagonist antibodies or antibody fragments.

The term “antibody” is used to mean an immunoglobulin molecule thatrecognizes and specifically binds to a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing etc., through at least one antigenrecognition site within the variable region of the immunoglobulinmolecule. As used herein, the term encompasses intact polyclonalantibodies, intact monoclonal antibodies, antibody fragments (such asFab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv) mutants,multispecific antibodies such as bispecific antibodies generated from atleast two intact antibodies, fusion proteins comprising an antibodyportion, and any other modified immunoglobulin molecule comprising anantigen recognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

As used herein, the term “antibody fragment” refers to a portion of anintact antibody and refers to the antigenic determining variable regionsof an intact antibody. Examples of antibody fragments include, but arenot limited to Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies,single chain antibodies, and multispecific antibodies formed fromantibody fragments.

An “Fv antibody” refers to the minimal antibody fragment that contains acomplete antigen-recognition and -binding site either as two-chains, inwhich one heavy and one light chain variable domain form a non-covalentdimer, or as a single-chain (scFv), in which one heavy and one lightchain variable domain are covalently linked by a flexible peptide linkerso that the two chains associate in a similar dimeric structure. In thisconfiguration the complementary determining regions (CDRs) of eachvariable domain interact to define the antigen-binding specificity ofthe Fv dimer. Alternatively a single variable domain (or half of an Fv)can be used to recognize and bind antigen, although generally with loweraffinity.

A “monoclonal antibody” as used herein refers to homogenous antibodypopulation involved in the highly specific recognition and binding of asingle antigenic determinant, or epitope. This is in contrast topolyclonal antibodies that typically include different antibodiesdirected against different antigenic determinants. The term “monoclonalantibody” encompasses both intact and full-length monoclonal antibodiesas well as antibody fragments (such as Fab, Fab′, F(ab′)2, Fv), singlechain (scFv) mutants, fusion proteins comprising an antibody portion,and any other modified immunoglobulin molecule comprising an antigenrecognition site. Furthermore, “monoclonal antibody” refers to suchantibodies made in any number of manners including, but not limited to,by hybridoma, phage selection, recombinant expression, and transgenicanimals.

As used herein, the term “humanized antibody” refers to forms ofnon-human (e.g., murine) antibodies that are specific immunoglobulinchains, chimeric immunoglobulins, or fragments thereof that containminimal non-human sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g. mouse, rat, rabbit, hamster, etc.) that have the desiredspecificity, affinity, and capability. In some instances, the Fvframework region (FR) residues of a human immunoglobulin are replacedwith the corresponding residues in an antibody from a non-human speciesthat has the desired specificity, affinity, and capability. Thehumanized antibody can be further modified by the substitution ofadditional residue either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539, hereinincorporated by reference.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-lazikani et al 1997, J. Molec.Bio. 273:927-948)). In addition, combinations of these two approachesare sometimes used in the art to determine CDRs.

The term “human antibody” as used herein means an antibody produced by ahuman or an antibody having an amino acid sequence corresponding to anantibody produced by a human made using any of the techniques known inthe art. This definition of a human antibody includes intact orfull-length antibodies, fragments thereof, and/or antibodies comprisingat least one human heavy and/or light chain polypeptide such as, forexample, an antibody comprising murine light chain and human heavy chainpolypeptides.

“Hybrid antibodies” are immunoglobulin molecules in which pairs of heavyand light chains from antibodies with different antigenic determinantregions are assembled together so that two different epitopes or twodifferent antigens can be recognized and bound by the resultingtetramer.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g. mouse, rat, rabbit, etc.) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

Competition between antibodies is determined by an assay in which theimmunoglobulin under study inhibits specific binding of a referenceantibody to a common antigen. Numerous types of competitive bindingassays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al., 1983,Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA(see Kirkland et al., J. Immunol. 1986, 137:3614-3619); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold SpringHarbor Press); solid phase direct label RIA using I-125 label (see Morelet al., 1988, Molec. Immunol. 25(1):7-15); solid phase directbiotin-avidin EIA (Cheung et al., 1990, Virology 176:546-552); anddirect labeled RA (Moldenhauer et al., 1990, Scand. J. Immunol.32:77-82). Typically, such an assay involves the use of purified antigenbound to a solid surface or cells bearing either of these, an unlabeledtest immunoglobulin and a labeled reference immunoglobulin. Competitiveinhibition is measured by determining the amount of label bound to thesolid surface or cells in the presence of the test immunoglobulin.Usually the test immunoglobulin is present in excess. Antibodiesidentified by competition assay (competing antibodies) includeantibodies binding to the same epitope as the reference antibody andantibodies binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antibody for steric hindrance to occur.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least50 or 75%.

That an antibody “selectively binds” or “specifically binds” to anepitope or receptor means that the antibody reacts or associates morefrequently, more rapidly, with greater duration, with greater affinity,or with some combination of the above to the epitope or receptor thanwith alternative substances, including unrelated proteins. “Selectivelybinds” or “specifically binds” means, for instance, that an antibodybinds to a protein with a K_(D) of about 0.1 mM or less, more usuallyabout 1 μM or less. “Selectively binds” or “specifically binds” means attimes that an antibody binds to a protein with a K_(D) of about 0.1 mMor less, at times about 1 μM or less, at times about 0.1 μM or less, attimes about 0.01 μM or less, and at times about 1 nM or less. Because ofthe sequence identity between homologous proteins in different species,specific binding can include an antibody that recognizes a Notchreceptor in more than one species. Likewise, because of homology betweendifferent Notch receptors (e.g., Notch2 and Notch3) in certain regionsof the polypeptide sequences of the receptors, specific binding caninclude an antibody that recognizes more than one Notch receptor. It isunderstood that, in certain embodiments, an antibody or binding moietythat specifically binds to a first target may or may not specificallybind to a second target. As such, “specific binding” does notnecessarily require (although it can include) exclusive binding, i.e.binding to a single target. Thus, an antibody may, in certainembodiments, specifically bind to more than one target (e.g., humanNotch2 and Notch3). In certain embodiments, the multiple targets may bebound by the same antigen-binding site on the antibody. For example, anantibody may, in certain instances, comprise two identicalantigen-binding sites, each of which specifically binds two or morehuman Notch receptors (e.g., human Notch2 and Notch3). In certainalternative embodiments, an antibody may be bispecific and comprise atleast two antigen-binding sites with differing specificities. By way ofnon-limiting example, a bispecific antibody may comprise oneantigen-binding site that recognizes an epitope on one Notch receptor,such as human Notch2, and further comprises a second, differentantigen-binding site that recognizes a different epitope on a secondNotch receptor, such as human Notch3. Generally, but not necessarily,reference to “binding” herein means “specific binding.”

As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

The terms “isolated” or “purified” refer to material that issubstantially or essentially free from components that normallyaccompany it in its native state. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A protein(e.g. an antibody) or nucleic acid that is the predominant speciespresent in a preparation is substantially purified. In particular, anisolated nucleic acid is separated from open reading frames thatnaturally flank the gene and encode proteins other than protein encodedby the gene. An isolated antibody is separated from othernon-immunoglobulin proteins and from other immunoglobulin proteins withdifferent antigen binding specificity. It can also mean that the nucleicacid or protein is at least 85% pure, at least 95% pure, and in someembodiments, at least 99% pure.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular examples of such cancers include squamouscell cancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer.

The terms “proliferative disorder” and “proliferative disease” refer todisorders associated with abnormal cell proliferation such as cancer.

“Tumor” and “neoplasm” as used herein refer to any mass of tissue thatresult from excessive cell growth or proliferation, either benign(noncancerous) or malignant (cancerous) including pre-cancerous lesions.

“Metastasis” as used herein refers to the process by which a cancerspreads or transfers from the site of origin to other regions of thebody with the development of a similar cancerous lesion at the newlocation. A “metastatic” or “metastasizing” cell is one that losesadhesive contacts with neighboring cells and migrates via thebloodstream or lymph from the primary site of disease to invadeneighboring body structures.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

The terms “cancer stem cell” or “tumor stem cell” or “solid tumor stemcell” are used interchangeably herein and refer to a population of cellsfrom a solid tumor that: (1) have extensive proliferative capacity; 2)are capable of asymmetric cell division to generate one or more kinds ofdifferentiated progeny with reduced proliferative or developmentalpotential; and (3) are capable of symmetric cell divisions forself-renewal or self-maintenance. These properties of “cancer stemcells” or “tumor stem cells” or “solid tumor stem cells” confer on thosecancer stein cells the ability to form palpable tumors upon serialtransplantation into an immunocompromised mouse compared to the majorityof tumor cells that fail to form tumors. Cancer stem cells undergoself-renewal versus differentiation in a chaotic manner to form tumorswith abnormal cell types that can change over time as mutations occur.

The terms “cancer cell” or “tumor cell” and grammatical equivalentsrefer to the total population of cells derived from a tumor includingboth non-tumorigenic cells, which comprise the bulk of the tumor cellpopulation, and tumorigenic stem cells (cancer stem cells).

As used herein “tumorigenic” refers to the functional features of asolid tumor stem cell including the properties of self-renewal (givingrise to additional tumorigenic cancer stem cells) and proliferation togenerate all other tumor cells (giving rise to differentiated and thusnon-tumorigenic tumor cells) that allow solid tumor stem cells to form atumor.

As used herein, the “tumorigenicity” of a tumor refers to the ability ofa random sample of cells from the tumor to form palpable tumors uponserial transplantation into immunocompromised mice.

As used herein, the terms “stein cell cancer marker” or “cancer stemcell marker” or “tumor stem cell marker” or “solid tumor stem cellmarker” refer to a gene or genes or a protein, polypeptide, or peptideexpressed by the gene or genes whose expression level, alone or incombination with other genes, is correlated with the presence oftumorigenic cancer cells compared to non-tumorigenic cells. Thecorrelation can relate to either an increased or decreased expression ofthe gene (e.g., increased or decreased levels of mRNA or the peptideencoded by the gene).

The terms “cancer stem cell gene signature” or “tumor stem cell genesignature” or “cancer stem cell signature” are used interchangeablyherein to refer to gene signatures comprising genes differentiallyexpressed in cancer stem cells compared to other cells or population ofcells, for example normal breast epithelial tissue. In some embodimentsthe cancer stem cell gene signatures comprise genes differentiallyexpressed in cancer stem cells versus normal breast epithelium by a foldchange, for example by 2 fold reduced and/or elevated expression, andfurther limited by using a statistical analysis such as, for example, bythe P value of a t-test across multiple samples. In another embodiment,the genes differentially expressed in cancer stem cells are divided intocancer stem cell gene signatures based on the correlation of theirexpression with a chosen gene in combination with their fold orpercentage expression change. Cancer stem cell signatures are predictiveboth retrospectively and prospectively of an aspect of clinicalvariability, including but not limited to metastasis and death.

The term “genetic test” as used herein refers to procedures whereby thegenetic make-up of a patient or a patient tumor sample is analyzed. Theanalysis can include detection of DNA, RNA, chromosomes, proteins ormetabolites to detect heritable or somatic disease-related genotypes orkaryotypes for clinical purposes.

As used herein, the terms “biopsy” or “biopsy tissue” refer to a sampleof tissue or fluid that is removed from a subject for the purpose ofdetermining if the sample contains cancerous tissue. In someembodiments, biopsy tissue or fluid is obtained because a subject issuspected of having cancer. The biopsy tissue or fluid is then examinedfor the presence or absence of cancer.

As used herein an “acceptable pharmaceutical carrier” refers to anymaterial that, when combined with an active ingredient of apharmaceutical composition such as an antibody, allows the antibody, forexample, to retain its biological activity. In addition, an “acceptablepharmaceutical carrier” does not trigger an immune response in arecipient subject. Examples include, but are not limited to, any of thestandard pharmaceutical carriers such as a phosphate buffered salinesolution, water, and various oil/water emulsions. Some diluents foraerosol or parenteral administration are phosphate buffered saline ornormal (0.9%) saline.

The term “therapeutically effective amount” refers to an amount of anantibody, polypeptide, polynucleotide, small organic molecule, or otherdrug effective to “treat” a disease or disorder in a subject or mammal.In the case of cancer, the therapeutically effective amount of the drugcan reduce the number of cancer cells; reduce the tumor size; inhibit orstop cancer cell infiltration into peripheral organs; inhibit and stoptumor metastasis; inhibit and stop tumor growth; relieve to some extentone or more of the symptoms associated with the cancer, or a combinationof such effects on cancer cells. To the extent the drug prevents growthand/or kills existing cancer cells, it can be referred to as cytostaticand/or cytotoxic.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent or slow the development of a targeted pathologiccondition or disorder. Thus those in need of treatment include thosealready with the disorder; those prone to have the disorder; and thosein whom the disorder is to be prevented. In some embodiments, a subjectis successfully “treated” for cancer according to the methods of thepresent invention if the patient shows one or more of the following: areduction in the number of or complete absence of cancer cells; areduction in the tumor size; inhibition of or an absence of cancer cellinfiltration into peripheral organs including the spread of cancer intosoft tissue and bone; inhibition of or an absence of tumor metastasis;inhibition or an absence of tumor growth; relief of one or more symptomsassociated with the specific cancer; reduced morbidity and mortality;and improvement in quality of life. Thus, in certain embodiments,treatment of cancer comprises inhibition of tumor growth in a subject.

As used herein, the terms “polynucleotide” or “nucleic acid” refer to apolymer composed of a multiplicity of nucleotide units (ribonucleotideor deoxyribonucleotide or related structural variants) linked viaphosphodiester bonds, including but not limited to, DNA or RNA. The termencompasses sequences that include any of the known base analogs of DNAand RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N-6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxyxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl 2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyl adenine,1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N6-methyladenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl 2-thiouracil,beta-D-mannosylqueosine, 5′-methoxycarbonylmethyluracil,5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyaceticacid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil,4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, and 2,6-diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length polypeptide or fragment areretained. The term also encompasses the coding region of a structuralgene and the sequences located adjacent to the coding region on both the5′ and 3′ ends for a distance of about 1 kb or more on either end suchthat the gene corresponds to the length of the full-length mRNA.Sequences located 5′ of the coding region and present on the mRNA arereferred to as 5′ non-translated sequences. Sequences located 3′ ordownstream of the coding region and present on the mRNA are referred toas 3′ non-translated sequences. The term “gene” encompasses both cDNAand genomic forms of a gene. A genomic form or clone of a gene containsthe coding region interrupted with non-coding sequences termed “introns”or “intervening regions” or “intervening sequences”. Introns aresegments of a gene that are transcribed into nuclear RNA (hnRNA);introns can contain regulatory elements such as enhancers. Introns areremoved or “spliced out” from the nuclear or primary transcript; intronstherefore are absent in the messenger RNA (mRNA) transcript. The mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide. In addition to containing introns,genomic forms of a gene can also include sequences located on both the5′ and 3′ end of the sequences that are present on the RNA transcript.These sequences are referred to as “flanking” sequences or regions(these flanking sequences are located 5′ or 3′ to the non-translatedsequences present on the mRNA transcript). The 5′ flanking region cancontain regulatory sequences such as promoters and enhancers thatcontrol or influence the transcription of the gene. The 3′ flankingregion can contain sequences that direct the termination oftranscription, post transcriptional cleavage, and polyadenylation.

The term “recombinant” when used with reference to a cell, nucleic acid,protein or vector indicates that the cell, nucleic acid, protein orvector has been modified by the introduction of a heterologous nucleicacid or protein, the alteration of a native nucleic acid or protein, orthat the cell is derived from a cell so modified. Thus, e.g.,recombinant cells express genes that are not found within the native(non-recombinant) form of the cell or express native genes that areoverexpressed or otherwise abnormally expressed such as, for example,expressed as non-naturally occurring fragments or splice variants. Bythe term “recombinant nucleic acid” herein is meant nucleic acid,originally formed in vitro, in general, by the manipulation of nucleicacid, e.g., using polymerases and endonucleases, in a form not normallyfound in nature. In this manner, operably linkage of different sequencesis achieved. Thus an isolated nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis invention. It is understood that once a recombinant nucleic acid ismade and introduced into a host cell or organism, it will replicatenon-recombinantly, i.e., using the in vivo cellular machinery of thehost cell rather than in vitro manipulations; however, such nucleicacids, once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention. Similarly, a “recombinant protein” is a protein madeusing recombinant techniques, i.e., through the expression of arecombinant nucleic acid as depicted above.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.” Vectorsare often derived from plasmids, bacteriophages, or plant or animalviruses.

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (e.g., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (e.g., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors”,respectively.

The terms “polypeptide” or “peptide” or “protein” or “protein fragment”are used interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refersto compounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an alpha carbon that is bound to a hydrogen,a carboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs can have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. “Amino acid variants” refers to amino acidsequences. With respect to particular nucleic acid sequences,conservatively modified variants refers to those nucleic acids whichencode identical or essentially identical amino acid sequences, or wherethe nucleic acid does not encode an amino acid sequence, to essentiallyidentical or associated (e.g., naturally contiguous) sequences. Becauseof the degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode most proteins. For instance, the codonsGCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at everyposition where an alanine is specified by a codon, the codon can bealtered to another of the corresponding codons described withoutaltering the encoded polypeptide. Such nucleic acid variations are“silent variations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes silent variations of the nucleic acid. It isrecognized that in certain contexts each codon in a nucleic acid (exceptAUG, which is ordinarily the only codon for methionine, and TGG, whichis ordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, silent variations of anucleic acid which encodes a polypeptide is implicit in a describedsequence with respect to the expression product, but not with respect toactual probe sequences. As to amino acid sequences, it will berecognized that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” includingwhere the alteration results in the substitution of an amino acid with achemically similar amino acid. Such conservatively modified variants arein addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention. Tables providing functionallysimilar amino acids useful for conservative amino acid substitutions arewell known in the art. Typical conservative substitutions include: 1)Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3)Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)). (See, also, Table 1 herein).

As used in the present disclosure and claims, the singular forms “a”,“an” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Certain Embodiments of the Present Invention

The present invention provides compositions and methods for studying,diagnosing, characterizing, and treating cancer. In particular, incertain embodiments, the present invention provides agents, includingantagonists, that bind Notch receptors and methods of using the agentsor antagonists to inhibit tumor growth and treat cancer or other diseasein human patients. In certain embodiments, the antagonists areantibodies that specifically recognize one or more human Notchreceptors.

In one aspect, the present invention provides an antibody thatspecifically binds to a non-ligand binding region of the extracellulardomain of a human Notch receptor. In some embodiments, the antibody thatspecifically binds to a non-ligand binding region of the extracellulardomain of a human Notch receptor inhibits growth of tumors. In certainembodiments, the antibody that specifically binds to a non-ligandbinding region of the extracellular domain of a human Notch receptor andinhibits tumor growth, specifically binds to a non-ligand binding regionof the extracellular domain of at least two Notch receptor familymembers. In certain embodiments, the antibody binds to a non-ligandbinding region of the extracellular domain of Notch2 and/or Notch3receptor. In some embodiments, the antibody binds to a non-ligandbinding region of the human Notch2. In some embodiments, the antibodybinds to a non-ligand binding region of the extracellular domain ofNotch2 and Notch3. In some embodiments, the antibody binds to anon-ligand binding region of the human Notch3. In some embodiments, theantibody binds to Notch1 and/or Notch4.

In certain embodiments, the antibody that specifically binds to anon-ligand binding region of the extracellular domain of a human Notchreceptor and inhibits tumor growth is a monoclonal antibody. In certainembodiments, the antibody that specifically binds to a non-ligandbinding region of the extracellular domain of a human Notch receptor andinhibits growth of tumors is a chimeric antibody. In certainembodiments, the antibody that specifically binds to a non-ligandbinding region of the extracellular domain of a human Notch receptor andinhibits growth of tumors is a humanized antibody. In certainembodiments, the antibody that specifically binds to a non-ligandbinding region of the extracellular domain of a human Notch receptor andinhibits tumor growth is a human antibody. In certain embodiments, theantibody that specifically binds to a non-ligand binding region of theextracellular domain of a human Notch receptor and inhibits tumor growthis a monospecific antibody. In certain embodiments, the antibody thatspecifically binds to a non-ligand binding region of the extracellulardomain of a human Notch receptor and inhibits tumor growth is abispecific antibody. In certain embodiments, the present inventionprovides a hybridoma producing an antibody that specifically binds to anon-ligand binding region of the extracellular domain of a human Notchreceptor and inhibits tumor growth.

In certain embodiments the present invention provides an antibody thatspecifically binds to a non-ligand binding region comprising EGF repeats1-10 of the extracellular domain of a human Notch receptor and inhibitstumor growth. In certain embodiments the present invention provides anantibody that specifically binds to a non-ligand binding regioncomprising EGF repeat 10 (or equivalent) of the extracellular domain ofa human Notch receptor and inhibits tumor growth. In certain embodimentsthe present invention provides an antibody that specifically binds to anon-ligand binding region comprising EGF repeats 13-36 of theextracellular domain of a human Notch receptor and inhibits tumorgrowth. Certain embodiments provide an antibody that specifically bindsto a non-ligand binding region comprising EGF repeats 4 of theextracellular domain of a human Notch receptor and inhibits tumorgrowth. Certain embodiments provide an antibody that specifically bindsto a non-ligand binding region comprising EGF repeat 13 of theextracellular domain of a human Notch receptor and inhibits tumorgrowth. In certain embodiments, the antibody specifically binds to anon-ligand binding region comprising the LNR-HD domain and inhibitstumor growth.

In certain embodiments the present invention provides a method oftreating cancer in a subject in need thereof comprising administering tothe subject a therapeutically effective amount of an antibody thatspecifically binds to a non-ligand binding region of the extracellulardomain of a human Notch receptor protein and inhibits tumor growth inthe subject. In certain embodiments, the method of treating cancercomprises administering a therapeutically effective amount of anantibody that specifically binds to at least two Notch receptor familymembers and inhibits tumor growth. In certain embodiments, the method oftreating cancer in a subject in need thereof comprises administering tothe subject a therapeutically effective amount of an antibody thatspecifically binds to a non-ligand binding region of the extracellulardomain of Notch2 and/or Notch3 receptor and inhibits tumor growth.

In certain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of a monoclonalantibody that specifically binds to a non-ligand binding region of theextracellular domain of a human Notch receptor and inhibits tumorgrowth. In certain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of a chimeric antibodythat specifically binds to a non-ligand binding region of theextracellular domain of a human Notch receptor and inhibits tumorgrowth. In certain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of a humanized antibodythat specifically binds to a non-ligand binding region of theextracellular domain of a human Notch receptor and inhibits tumorgrowth. In certain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of a human antibodythat specifically binds to a non-ligand binding region of theextracellular domain of a human Notch receptor and inhibits tumorgrowth. In some embodiments, the antibody is a monospecific antibody. Insome embodiments, the antibody is a bispecific antibody.

In certain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of an antibody thatspecifically binds to a non-ligand binding region of the extracellulardomain of a human Notch receptor comprising EGF repeats 1-10 andinhibits tumor growth. In certain embodiments, the method of treatingcancer comprises administering a therapeutically effective amount of anantibody that specifically binds to a non-ligand binding region of theextracellular domain of a human Notch receptor comprising EGF repeat 10(or the equivalent if Notch3) and inhibits tumor growth. In certainembodiments, the method of treating cancer comprises administering atherapeutically effective amount of an antibody that specifically bindsto a non-ligand binding region of the extracellular domain of a humanNotch receptor comprising EGF repeats 13-36 and inhibits tumor growth.In certain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of an antibody thatspecifically binds to a non-ligand binding region of the extracellulardomain of a human Notch receptor comprising EGF repeat 4 and inhibitstumor growth. In certain embodiments, the method of treating cancercomprises administering a therapeutically effective amount of anantibody that specifically binds to a non-ligand binding region of theextracellular domain of a human Notch receptor comprising EGF repeat 4and inhibits tumor growth. In certain other embodiments, the antibodythat is administered specifically binds to the LNR-HD domain of a humanNotch receptor.

In certain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of an antibodyconjugated to a cytotoxic moiety that specifically binds to a non-ligandbinding region of the extracellular domain of a human Notch receptor andinhibits tumor growth. In certain embodiments, the method of treatingcancer comprises administering a therapeutically effective amount of anantibody that specifically binds to a non-ligand binding region of theextracellular domain of a human Notch receptor and inhibits tumor growthin combination with radiation therapy. In certain embodiments, themethod of treating cancer comprises administering a therapeuticallyeffective amount of an antibody that specifically binds to a non-ligandbinding region of the extracellular domain of a human Notch receptor andinhibits tumor growth in combination with chemotherapy. In certainembodiments, the method of treating cancer comprises administering atherapeutically effective amount of an antibody that specifically bindsto a non-ligand binding region of the extracellular domain of a humanNotch receptor and inhibits tumor growth that are from a breast tumor,colorectal tumor, lung tumor, pancreatic tumor, prostate tumor, or ahead and neck tumor.

In certain embodiments, the method of treating cancer comprisesidentifying patients for treatment with the antibody that specificallybinds to a non-ligand binding region of the extracellular domain of ahuman Notch receptor using a genetic test; and administering atherapeutically effective amount of an antibody that specifically bindsto a non-ligand binding region of the extracellular domain of a humanNotch receptor and inhibits tumor growth. In certain embodiments, themethod of treating cancer comprises identifying patients for treatmentwith the antibody that specifically binds to a non-ligand binding regionof the extracellular domain of a human Notch receptor using a genetictest that detects a cancer stem cell signature; and administering atherapeutically effective amount of an antibody that specifically bindsto a non-ligand binding region of the extracellular domain of a humanNotch receptor and inhibits tumor growth.

In certain embodiments, the present invention provides a method ofidentifying a molecule that binds to a non-ligand binding region of anextracellular domain of a human Notch receptor and inhibits tumorgrowth, the method comprising: i) incubating the molecule with thenon-ligand binding domain of the extracellular domain of a human Notchreceptor; ii) determining if the molecule binds to the non-ligandbinding region of the extracellular domain of the human Notch receptor;and iii) determining if the molecule inhibits tumor growth. In certainembodiments, the invention provides a method of identifying a moleculethat binds to a non-ligand binding region of an extracellular domain ofa human Notch receptor and inhibits tumor growth, the method comprising:i) incubating the molecule with the non-ligand binding domain of theextracellular domain of a human Notch receptor comprising EGF repeats1-10; ii) determining if the molecule binds to the non-ligand bindingregion of the extracellular domain of the human Notch receptorcomprising EGF repeats 1-10; and iii) determining if the moleculeinhibits tumor growth. In certain embodiments, the invention provides amethod of identifying a molecule that binds to a non-ligand bindingregion of an extracellular domain of a human Notch receptor and inhibitstumor growth, the method comprising: i) incubating the molecule with thenon-ligand binding domain of the extracellular domain of a human Notchreceptor comprising EGF repeat 10 (or equivalent if Notch3); ii)determining if the molecule binds to the non-ligand binding region ofthe extracellular domain of the human Notch receptor comprising EGFrepeat 10 (or equivalent if Notch3); and iii) determining if themolecule inhibits tumor growth. In certain embodiments, the inventionprovides a method of identifying a molecule that binds to a non-ligandbinding region of an extracellular domain of a human Notch receptor andinhibits tumor growth, the method comprising: i) incubating the moleculewith the non-ligand binding domain of the extracellular domain of ahuman Notch receptor comprising EGF repeats 13-36; ii) determining ifthe molecule binds to the non-ligand binding region of the extracellulardomain of the human Notch receptor comprising EGF repeats 13-36; andiii) determining if the molecule inhibits tumor growth.

In certain embodiments, the present invention provides a pharmaceuticalcomposition comprising an antibody that specifically binds to anon-ligand binding region of the extracellular domain of a human Notchreceptor and inhibits tumor growth.

in certain embodiments, the present invention provides a method ofmaking an antibody that specifically binds to a non-ligand bindingregion of the extracellular domain of a human Notch receptor andinhibits tumor growth.

In certain embodiments, the present invention provides an isolatednucleic acid that encodes an antibody that specifically binds to anon-ligand binding region of the extracellular domain of a human Notchreceptor and inhibits tumor growth.

In some embodiments, the invention provides an agent (e.g., an antibody)that specifically binds to an EGF10 domain (or an equivalent of an EGF10domain if Notch3) of one or more human Notch receptors. In certainembodiments, the agent is an antibody. In certain embodiments, the agentis an antagonist. In certain embodiments, the agent specifically bindsto EGF10 of human Notch2 and/or EGF9 of human Notch3. EGF9 is the EGFwithin human Notch3 that is equivalent to EGF10 in the other human Notchreceptors Notch1, Notch2, and Notch4. In certain embodiments, the agentspecifically binds human Notch2. In certain embodiments, the agentspecifically binds human Notch2 and Notch3. In certain embodiments, theagent specifically binds human Notch3.

In one aspect, the invention provides a 59R1 antibody comprising theheavy chain and light chain sequences provided in SEQ ID NOs:16 and 18(with or without signal sequence), respectively, or as encoded by theDNA deposited with ATCC on Oct. 15, 2008, and assigned designationnumber PTA-9547. The invention further provides polypeptides orantibodies that comprise the heavy chain variable region (e.g., SEQ IDNO: 14) and/or the light chain variable region (e.g., SEQ ID NO:13) ofsuch a 59R1 antibody. The invention further provides polypeptides orantibodies comprising one or more (e.g., 1, 2, or 3) of the heavy chainCDRs, and/or one or more of the light chain CDRs of the 59R1 antibody.In still further embodiments, the invention provides antibodies thatbind to the same epitope as the 59R1 antibody or antibodies that competefor specific binding to human Notch2 and/or Notch3 with the 59R1antibody.

In another aspect, the invention provides a 59R5 antibody comprising theheavy chain and light chain sequences provided in SEQ ID NOs:49 and 18(with or without signal sequence), respectively, or as encoded by theDNA deposited with ATCC on Jul. 6, 2009 and assigned designation numberPTA-10170. The invention further provides polypeptides or antibodiesthat comprise the heavy chain variable region and/or the light chainvariable region sequences SEQ ID NO:50 and/or SEQ ID NO:13. Theinvention further provides polypeptides or antibodies comprising one ormore (e.g., 1, 2, or 3) of the heavy chain CDRs and/or one or more ofthe light chain CDRs of the 59R5 antibody. In still further embodiments,the invention provides antibodies that bind to the same epitope as the59R5 antibody or antibodies that compete for specific binding to humanNotch2 and/or Notch3 with the 59R5 antibody.

In certain additional embodiments, the invention provides an antibodythat specifically binds to two or more (i.e., at least two or two,three, or four) human Notch receptors. In certain embodiments, theantibody specifically binds to a non-ligand binding region of anextracellular domain of the two or more human Notch receptors. Incertain embodiments, the antibody is a monospecific antibody thatspecifically binds to a non-ligand binding region of an extracellulardomain of the two or more human Notch receptors. In certain embodiments,the antibody binds to EGF10 of Notch1, Notch2, or Notch4, and/or to EGF9of Notch3. In certain embodiments, the non-ligand binding region towhich the antibody binds is not EGF4 or does not comprise EGF4. Incertain embodiments, the two or more human Notch receptors compriseNotch2 and/or Notch3. In certain embodiments, the two or more humanNotch receptors comprise Notch2 and Notch3. In certain embodiments, theantibody is an antagonist of the two or more human Notch receptors.

The invention further provides a method of modulating the function ofpericytes and/or vascular smooth muscle cells in a subject, wherein themethod comprises administering to the subject an effective amount of anagent that specifically binds human Notch2 and/or human Notch3. Incertain embodiments, the agent is an antibody. In certain embodiments,the agent is an antagonist.

The invention further provides a method of inhibiting angiogenesis in asubject, comprising the step of administering to the subject aneffective amount of an agent that specifically binds human Notch2 and/orhuman Notch3. In certain embodiments, the agent is an antibody. Incertain embodiments, the agent is an antagonist. In certain embodiments,the antagonist is an antagonist of Notch2. In certain embodiments, theantagonist is an antagonist of Notch3. In certain embodiments, theantagonist is an antagonist of Notch2 and Notch3. In some embodiments,the method of inhibiting angiogenesis comprises modulating the functionof pericytes and/or vascular smooth muscle cells. In some embodiments,the angiogenesis is tumor angiogenesis.

In certain embodiments, the Notch-binding agent is an antagonist of thehuman Notch receptor(s) to which it specifically binds. In somealternative embodiments, the Notch-binding agent is an agonist of thehuman Notch receptor(s) to which it specifically binds.

In certain embodiments, the agent that specifically binds to one or moreNotch receptor(s) and is an antagonist of the one or more Notchreceptor(s) inhibits at least about 10%, at least about 20%, at leastabout 30%, at least about 50%, at least about 75%, at least about 90%,or about 100% of one or more activities of the bound Notch receptor(s).

In certain embodiments, the antagonist of one or more human Notchreceptor(s) (e.g., Notch2 and/or Notch3) has one or more of thefollowing effects: inhibit ligand binding to the one or more human Notchreceptors, inhibit ligand-induced signaling by the one or more Notchreceptors; inhibit proliferation of tumor cells; reduce thetumorigenicity of a tumor by reducing the frequency of cancer steincells in the tumor; inhibit tumor growth; increase survival, triggercell death of tumor cells; inhibit angiogenesis; or prevent metastasisof tumor cells.

In certain embodiments, the antagonist has one or more of the followingeffects: interference with the expression of a Notch receptor;interference with activation of a Notch receptor signal transductionpathway by, for example, sterically inhibiting interactions between theNotch receptor and one or more of its ligands, or binding to a humanNotch receptor and triggering cell death or inhibiting cellproliferation.

In certain embodiments, antagonists against a Notch receptor, such asNotch2 or Notch3, act extracellularly to act upon or inhibit thefunction of the Notch receptor. In certain embodiments, an antagonist isa small molecule that binds to the extracellular domain of a Notchreceptor. In certain embodiments, an antagonist of a Notch receptor isproteinaceous. In some embodiments, proteinaceous antagonists of a Notchreceptor are antibodies that specifically bind to an extracellularepitope of a Notch receptor. Extracellular binding of an antagonistagainst a Notch receptor can inhibit the signaling of a Notch receptorprotein by inhibiting intrinsic activation (e.g., kinase activity) of aNotch receptor and/or by sterically inhibiting the interaction, forexample, of a Notch receptor with one of its ligands. Furthermore,extracellular binding of an antagonist against a Notch receptor candown-regulate cell-surface expression of a Notch receptor such as, forexample, by internalization of a Notch receptor and/or decreasing cellsurface trafficking of a Notch receptor.

In certain embodiments, the Notch-binding agent or antagonist (e.g.,antibody) specifically binds to a non-ligand binding region of anextracellular domain of at least one human Notch receptor, wherein thenon-ligand binding region comprises EGF repeat 10 (or the equivalent ifNotch3). In certain embodiments, the agent or antagonist specificallybinds to Notch2. In certain embodiments, the agent or antagonistspecifically binds to Notch3. In certain embodiments, the agent orantagonist specifically binds to both human Notch2 and human Notch3.

In certain embodiments, the Notch-binding agent or antagonist (e.g.,antibody) specifically binds to an EGF10 domain of human Notch2. Incertain embodiments, the Notch-binding agent or antagonist does not bindto any region of the human Notch2 outside of the EGF10 domain. Incertain alternative embodiments, the Notch-binding agent or antagonistthat specifically binds to an EGF10 domain of human Notch2, also furtherbinds to another region of human Notch2. In other words, in someembodiments, the entire epitope of the agent or antagonist falls withinEGF10. In certain other embodiments, the epitope of the agent orantagonist that binds to human Notch2 partially overlaps with EGF10. Incertain embodiments, the agent or antagonist binds to at least part ofthe sequence HKGAL (SEQ ID NO:28) within human Notch2 EGF10. In certainembodiments, the agent or antagonist also binds to other amino acidswithin human Notch2 EGF10 (e.g., the entire epitope of an anti-Notch2antibody is not necessarily contained entirely within the sequenceHKGAL). In certain embodiments, the Notch-binding agent or antagonistfurther specifically binds to at least one additional human Notchreceptor (e.g., Notch1, Notch3, or Notch4). In certain embodiments, theNotch-binding agent or antagonist that binds to EGF10 of human Notch2further binds to an EGF10 domain of human Notch 1, an EGF9 domain ofhuman Notch3, and/or an EGF 10 domain of human Notch4. In certainembodiments, the additional human Notch receptor is human Notch3.

In certain embodiments, the Notch-binding agent or antagonist (e.g.,antibody) binds to an EGF9 domain of human Notch3. As is apparent fromthe homology between the sequences of the extracellular domains of humanNotch2 and human Notch3, EGF9 is the EGF that is thefunctional/structural equivalent of Notch2 EGF10 in human Notch3. Incertain embodiments, the Notch-binding agent or antagonist does not bindto any region of the human Notch3 outside of EGF9. In certainembodiments, the agent or antagonist binds to at least part of thesequence HEDAI (SEQ ID NO:29) within the human Notch3 EGF9 domain. HEDAI(SEQ ID NO:29) is the sequence within the Notch3 EGF9 domain thatcorresponds to sequence HKGAL (SEQ ID NO:28) within human Notch2 EGF10.In certain embodiments, the agent or antagonist also binds to otheramino acids within human Notch3 EGF9. In certain embodiments, theNotch-binding agent or antagonist binds to an EGF10 domain of at leastone additional human Notch receptor (e.g., Notch1, Notch2, and/orNotch4). In certain embodiments, the additional human Notch receptor ishuman Notch2, such as an agent or antagonist that binds to an EGF10domain of human Notch2. In certain embodiments, the Notch-binding agentor antagonist does not bind to any region of the human Notch2 outside ofEGF10. In certain embodiments, the agent or antagonist binds to at leastpart of the sequence HKGAL (SEQ ID NO:28) within human Notch2 EGF10. Insome embodiments, the agent or antagonist is a monospecific antibodythat binds to at least part of the sequence HKGAL (SEQ ID NO:28) inNotch2 and also binds to at least part of the sequence HEDAI (SEQ IDNO:29) in Notch3.

In certain alternative embodiments, the Notch-binding agent orantagonist binds to a portion of the non-ligand binding region of anextracellular domain of a Notch1, Notch2, or Notch4 receptor in a regionother than EGF10 or a Notch3 receptor in a region other than EGF9. Forexample, in certain embodiments, the agent or antagonist binds to theLNR-1-ID domain of one or more Notch receptors. In certain embodiments,the agent or antagonist binds to EGF1, EGF2, EGF3, EGF4, EGF5, EGF6,EGF7, EGF9, EGF10, EGF13, EGF14, EGF15, EGF16, EGF17, EGF18, EGF19,EGF20, EGF21, EGF22, EGF23, EGF24, EGF25, EGF26, EGF27, EGF28, EGF29,EGF30, EGF31, EGF32, EGF33, EGF34, EGF35, and/or EGF36 of anextracellular domain of one or more Notch receptors.

In certain embodiments, the Notch-binding agent or antagonist binds tothe ligand binding region of an extracellular domain of one or morehuman Notch receptors. Thus, in certain embodiments, the Notch-bindingagent or antagonist may bind to EGF11 and/or EGF12 of Notch 1, 2, or 4(Rebay et al., 1991, Cell 67:687; Lei et al., 2003, Dev. 130:6411;Hambleton et al., 2004, Structure 12:2173) or EGF10 and/or EGF11 ofNotch3 (Peters et al., 2004, Experimental Cell Research, 299:454-464).

In certain embodiments, the Notch-binding agent (e.g., antibody)specifically binds to two or more human Notch receptors (e.g., Notch1,Notch2, Notch3, and/or Notch4). In other words, in certain embodiments,the agent or antibody binds at least two human Notch receptors (i.e.,two, three, or four human Notch receptors). Encompassed are agents andantibodies that specifically bind to two human Notch family receptors(e.g., Notch2 and Notch3, Notch1 and Notch2, Notch1 and Notch3, Notch1and Notch4, Notch2 and Notch4, or Notch3 and Notch4). Agents andantibodies that specifically bind to three human Notch receptor familymembers are also envisioned (e.g., agents and antibodies thatspecifically bind to Notch1, Notch2, and Notch3, Notch1, Notch2, andNotch4, or Notch2, Notch3, and Notch4), as are agents and antibodiesthat specifically bind to four human Notch receptor family members(e.g., agents and antibodies that specifically bind to Notch1, Notch2,Notch3 and Notch4). In certain embodiments, the agent or antibodyspecifically binds to both human Notch2 and Notch3. In certainalternative embodiments, the agent or antibody specifically binds toboth human Notch1 and Notch2. In some embodiments, the agent or antibodyspecifically binds to both human Notch1 and Notch3. In still furtherembodiments, the agent or antibody specifically binds to both humanNotch1 and Notch4. In certain embodiments, the agent or antibody is anantagonist of the two or more human Notch receptors.

In certain embodiments, the Notch-binding agent or antagonist binds to aNotch receptor (e.g., Notch2 and/or Notch3) with a dissociation constantof about 1 μM or less, about 100 nM or less, about 40 nM or less, about20 nM or less, or about 10 nM or less. In certain embodiments, the agentor antagonist binds one or more human Notch receptors, such as humanNotch2 and/or human Notch3, with a K_(D) of 1 nM or less. In someembodiments, the Notch binding agent is an antibody that binds to Notch2with a K_(D) of about 1 nM or less. In some embodiments, the Notchbinding agent is an antibody that binds to Notch3 with a K of about 1 nMor less. In certain embodiments, the dissociation constant for the agentor antagonist with respect to a particular Notch receptor is thedissociation constant determined using a Notch-Fc fusion proteincomprising the Notch extracellular domain and/or a portion of theextracellular domain comprising EGF10 immobilized on a Biacore chip.

In certain embodiments, the antagonist specifically binds to humanNotch3 and inhibits binding of a ligand (e.g., DLL4, JAG1, and/or JAG2)to human Notch3 and/or inhibits signaling of human Notch3. In certainembodiments, the antagonist specifically binds to human Notch2 andinhibits binding of a ligand (e.g., DLL4, JAG1, and/or JAG2) to humanNotch2 and/or inhibits signaling of human Notch2. In certainembodiments, the antagonist inhibits DLL4-induced Notch2 signaling. Incertain embodiments, the antagonist inhibits DLL4-induced Notch3signaling. In certain embodiments, the antagonist inhibits JAG2-inducedNotch2 signaling. In certain embodiments, the antagonist inhibitsJAG2-induced Notch3 signaling. In certain embodiments, the signaling byNotch2 and/or Notch3 is reduced by at least about 10%, by at least about25%, by at least about 50%, by at least about 75%, by at least about 90%or by at least about 95%. In certain embodiments, the binding of one ormore ligands to Notch2 and/or Notch3 is reduced by at least about 10%,by at least about 25%, by at least about 50%, by at least about 75%, byat least about 90%, or by at least about 95%.

In some embodiments, antagonists against a Notch receptor bind to aNotch receptor and have one or more of the following effects: inhibitproliferation of tumor cells, trigger cell death directly in tumorcells, or prevent metastasis of tumor cells. In certain embodiments,antagonists of a Notch receptor trigger cell death via a conjugatedtoxin, chemotherapeutic agent, radioisotope, or other such agent. Forexample, an antibody against a Notch receptor is conjugated to a toxinthat is activated in tumor cells expressing the Notch receptor byprotein internalization. In other embodiments, antagonists of a Notchreceptor mediate cell death of a cell expressing the Notch receptor viaantibody-dependent cellular cytotoxicity (ADCC). ADCC involves celllysis by effector cells that recognize the Fc portion of an antibody.Many lymphocytes, monocytes, tissue macrophages, granulocytes andeosinophils, for example, have Fe receptors and can mediate cytolysis(Dillman, 1994, J. Clin. Oncol. 12:1497). In some embodiments, anantagonist of a Notch receptor is an antibody that triggers cell deathof cell expressing a Notch receptor by activating complement-dependentcytotoxicity (CDC). CDC involves binding of serum complement to the Fcportion an antibody and subsequent activation of the complement proteincascade, resulting in cell membrane damage and eventual cell death.Biological activity of antibodies is known to be determined, to a largeextent, by the constant domains or Fc region of the antibody molecule(Uananue and Benacerraf, Textbook of Immunology, 2nd Edition, Williams &Wilkins, p. 218 (1984)). Antibodies of different classes and subclassesdiffer in this respect, as do antibodies of the same subclass but fromdifferent species. Of human antibodies, IgM is the most efficient classof antibodies to bind complement, followed by IgG1, IgG3, and IgG2whereas IgG4 appears quite deficient in activating the complementcascade (Dillman, 1994, J. Clin. Oncol. 12:1497; Jefferis et al., 1998,Immunol. Rev. 163:59-76). According to the present invention, antibodiesof those classes having the desired biological activity are prepared.

The ability of any particular antibody against a Notch receptor tomediate lysis of the target cell by complement activation and/or ADCCcan be assayed. The cells of interest are grown and labeled in vitro;the antibody is added to the cell culture in combination with eitherserum complement or immune cells which can be activated by the antigenantibody complexes. Cytolysis of the target cells is detected, forexample, by the release of label from the lysed cells. In fact,antibodies can be screened using the patient's own serum as a source ofcomplement and/or immune cells. The antibody that is capable ofactivating complement or mediating ADCC in the in vitro test can then beused therapeutically in that particular patient.

In certain embodiments, the Notch-binding agent or antagonist is anantibody that does not have one or more effector functions. Forinstance, in some embodiments, the antibody has no antibody-dependentcellular cytoxicity (ADCC) activity and/or no complement-dependentcytoxicity (CDC) activity. In certain embodiments, the antibody does notbind to an Fc receptor and/or complement factors. In certainembodiments, the antibody has no effector function.

In other embodiments, antagonists of a Notch receptor can trigger celldeath indirectly by inhibiting angiogenesis. Angiogenesis is the processby which new blood vessels form from pre-existing vessels and is afundamental process required for normal growth, for example, duringembryonic development, wound healing, and in response to ovulation.Solid tumor growth larger than 1-2 mm² also requires angiogenesis tosupply nutrients and oxygen without which tumor cells die. Thus, incertain embodiments, an antagonist of a Notch receptor targets vascularcells that express the Notch receptor including, for example,endothelial cells, smooth muscle cells or components of theextracellular matrix required for vascular assembly. In certainembodiments, an antagonist of a Notch receptor (e.g., Notch2 and/orNotch3) targets pericytes and/or vascular smooth muscle cells. In otherembodiments, an antagonist of a Notch receptor inhibits growth factorsignaling required by vascular cell recruitment, assembly, maintenanceor survival. In certain embodiments, the antagonist modulates thefunction of pericytes and/or vascular smooth muscle cells.

In certain embodiments the Notch-binding agents or antagonists (e.g.,antibodies), either alone or in combination with a second therapeuticagent, are capable of inhibiting tumor growth. In certain embodiments,the Notch-binding agents or antagonists are capable of inhibiting tumorgrowth in vivo (e.g., in a xenograft mouse model and/or in a humanhaving cancer). In certain embodiments, the Notch-binding agents orantagonists are capable of inhibiting tumor growth by at least about10%, at least about 25%, at least about 50%, at least about 75%, atleast about 90% at a given time point in a xenograft model. In certainembodiments, the Notch-binding agents or antagonists prevent tumorgrowth. In certain embodiments, the Notch-binding agents or antagonistsinhibit tumor recurrence.

In certain embodiments, the Notch-binding agents are capable of reducingthe tumorigenicity of a tumor. In certain embodiments, the agent orantibody is capable of reducing the tumorigenicity of a tumor comprisingcancer stem cells in an animal model, such as a mouse xenograft model.In certain embodiments, the number or frequency of cancer stem cells ina tumor is reduced by at least about two-fold, about three-fold, aboutfive-fold, about ten-fold, about 50-fold, about 100-fold, or about1000-fold (e.g., in a xenograft model). In certain embodiments, thereduction in the frequency of cancer stem cells is determined bylimiting dilution assay using an animal model. An example of a limitingdilution assay used to test the efficacy of an anti-Notch antibody isprovided in Example 8, below. Additional examples and guidance regardingthe use of limiting dilution assays to determine a reduction in thenumber or frequency of cancer stem cells in a tumor can be found, e.g.,in international Publication No. WO 2008/042236, U.S. Patent ApplicationPublication Nos. 2008/0064049, and 2008/0178305, each of which isincorporated by reference herein in its entirety.

The present invention provides a variety of polypeptides, including butnot limited to, antibodies and fragments of antibodies. In certainembodiments, the polypeptide is isolated. In certain alternativeembodiments, the polypeptide is substantially pure.

In certain embodiments, the polypeptides of the present invention can berecombinant polypeptides, natural polypeptides, or syntheticpolypeptides comprising the sequence of SEQ ID NOs:2, 4, 13, 14, 16, 18,19, 20, 39, 40, 49, 50, 52, 53, 54, 55, 56, or 57 (with or without theindicated signal sequences), as well as the polypeptides comprising thepolypeptides encoded by the polynucleotides of SEQ ID NOs: 1, 3, 15, 17,47, 48, 58, 59, or 60 (with or without the indicated signal sequences).

The invention provides a polypeptide comprising the heavy chain and/orthe light chain of 59R1 provided in SEQ ID NO:16 and/or SEQ ID NO:18,respectively. In certain embodiments, the polypeptide is an antibody. Incertain embodiments, the polypeptide specifically binds Notch2 and/orNotch3. In some embodiments, the polypeptide specifically binds Notch2and Notch3.

The invention provides a polypeptide comprising the heavy chain and/orthe light chain of 59R5 provided in SEQ ID NO:49 and/or SEQ ID NO:18,respectively. In certain embodiments, the polypeptide is an antibody. Incertain embodiments, the polypeptide specifically binds Notch2 and/orNotch3. In some embodiments, the polypeptide specifically binds Notch2and Notch3.

The invention further provides a polypeptide comprising SEQ ID NO:13and/or SEQ ID NO:14. In certain embodiments, the polypeptide comprises avariable light chain sequence comprising SEQ ID NO:13 and/or a variableheavy chain sequence comprising SEQ ID NO:14. In some embodiments, thepolypeptide comprises a variable light chain sequence comprising SEQ IDNO:13 and a variable heavy chain sequence comprising SEQ ID NO:14. Incertain embodiments, the polypeptide comprises a variable light chainsequence comprising SEQ ID NO:13 and/or a variable heavy chain sequencecomprising SEQ ID NO:50. In some embodiments, the polypeptide comprisesa variable light chain sequence comprising SEQ ID NO:13 and a variableheavy chain sequence comprising SEQ ID NO:50. In certain embodiments,the polypeptide comprises a variable light chain sequence comprising SEQID NO:13 and/or a variable heavy chain sequence comprising SEQ ID NO:52.In certain embodiments, the polypeptide comprises a variable light chainsequence comprising SEQ ID NO:13 and/or a variable heavy chain sequencecomprising SEQ ID NO:53. In certain embodiments, the polypeptidecomprises a variable light chain sequence comprising SEQ ID NO:13 and/ora variable heavy chain sequence comprising SEQ ID NO:54. In certainembodiments, the polypeptide comprises a variable light chain sequencecomprising SEQ ID NO:13 and/or a variable heavy chain sequencecomprising SEQ ID NO:55. In certain embodiments, the polypeptidecomprises a variable light chain sequence comprising SEQ ID NO:13 and/ora variable heavy chain sequence comprising SEQ ID NO:56. In certainembodiments, the polypeptide comprises a variable light chain sequencecomprising SEQ ID NO:13 and/or a variable heavy chain sequencecomprising SEQ ID NO:57. In certain embodiments, the polypeptide is anantibody. In certain embodiments, the polypeptide specifically bindsNotch2 and/or Notch3. In some embodiments, the polypeptide specificallybinds Notch2 and Notch3. In some embodiments, the polypeptidespecifically binds human Notch2. In some embodiments, the polypeptidespecifically binds human Notch3.

It will be recognized in the art that some amino acid sequences of theinvention can be varied without significant effect of the structure orfunction of the protein. If such differences in sequence arecontemplated, it should be remembered that there will be critical areason the protein which determine activity. Thus, the invention furtherincludes variations of the polypeptides which show substantial activity.Such mutants include deletions, insertions, inversions, repeats, andtype substitutions. Guidance concerning which amino acid changes arelikely to be phenotypically silent can be found in Bowie et al.,Deciphering the Message in Protein Sequences Tolerance to Amino AcidSubstitutions, 1990, Science 247:1306-1310.

Thus, the fragments, derivatives, or analogs of the polypeptides of theinvention can be: (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (often a conserved amino acid residue) and such substitutedamino acid residue can or cannot be one encoded by the genetic code; or(ii) one in which one or more of the amino acid residues includes asubstituent group; or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol); or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives, and analogs are deemed to be within the scope ofthe teachings herein.

Of particular interest are substitutions of a charged amino acid withanother charged amino acid and with neutral or negatively charged aminoacid. The latter results in proteins with reduced positive charge.Reduced positive charge on a protein can lead to reduction in proteinaggregation and the prevention of aggregation is highly desirable.Aggregation of proteins can not only result in a loss of activity butcan also be problematic when preparing pharmaceutical formulations,because aggregates can be immunogenic. (Pinckard et al., 1967, Clin.Exp. Immunol. 2:331-340; Robbins et al., 1987, Diabetes 36:838-845;Cleland et al., 1993, Crit. Rev. Therapeutic Drug Carrier Systems10:307-377).

As indicated, amino acid changes are typically of a minor nature, suchas conservative amino acid substitutions that do not significantlyaffect the folding or activity of the protein (see Table 1.)

TABLE 1 Conservative Amino Acid Substitutions Original Amino ExemplaryConservative Acid Substitutions Alanine Valine, Isoleucine, Leucine,Glycine, Serine Arginine Lysine, Histidine, Glutamine, AsparagineAsparagine Glutamine, Histidine, Lysine, Arginine Aspartic Acid GlutamicAcid, Asparagine Cysteine Serine, Alanine, Methionine GlutamineAsparagine Glutamic Acid Aspartic Acid, Glutamine Glycine Proline,Alanine Histidine Asparagine, Glutamine, Lysine, Arginine IsoleucineLeucine, Valine, Methionine, Alanine, Phenylalanine, Norleucine LeucineNorleucine, Isoleucine, Valine, Methionine, Alanine, PhenylalanineLysine Arginine, Glutamine, Asparagine, Histidine Methionine Leucine,Phenylalanine, Isoleucine, Valine, Cysteine Phenylalanine Leucine,Valine, Isoleucine, Alanine, Tyrosine Proline Alanine, Glycine SerineThreonine Threonine Serine Trytophan Tyrosine, Phenylalanine TyrosineTryptophan, Phenylalanine, Threonine, Serine Valine Isoleucine,Methionine, Leucine, Phenylalanine, Alanine, Norleucine

Of course, the number of amino acid substitutions made depends on manyfactors, including those described above. In certain embodiments, thenumber of substitutions for any given polypeptide will not be more than50, 40, 30, 25, 20, 15, 10, or 3.

In certain embodiment, the polypeptides and polynucleotides of thepresent invention are provided in an isolated form, and at times arepurified to homogeneity.

The polypeptides of the present invention include the polypeptides ofSEQ ID NOs: 2, 4, 13, 14, 16, 18, 19, 20, 39, 40, 49, 50, 52, 53 54, 55,56, or 57 as well as polypeptides which have at least 90% similarity (atcertain times at least 90% sequence identity) to the polypeptides of SEQID NOs: 2, 4, 13, 14, 16, 18, 19, 20, 39, 40, 49, 50, 52, 53 54, 55, 56,or 57 and at least 95% similarity (at certain times at least 95%sequence identity) to the polypeptides of SEQ ID NOs: 2, 4, 13, 14, 16,18, 19, 20, 49, 50, 52, 53 54, 55, 56, or 57 and in still otherembodiments, polypeptide which have at least 96%, 97%, 98%, or 99%similarity (at certain times 96%, 97%, 98%, or 99% sequence identity) tothe polypeptides of SEQ ID NOs: 2, 4, 13, 14, 16, 18, 19, 20, 39, 40,49, 50, 52, 53 54, 55, 56, or 57. As known in the art, “similarity”between two polypeptides is determined by comparing the amino acidsequence and its conserved amino acid substitutes of one polypeptide tothe sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention canbe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments can be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention can be used tosynthesize full-length polynucleotides of the present invention.

In certain embodiments, a fragment of the proteins of this invention isa portion or all of a protein which is capable of binding to a Notchreceptor protein. This fragment has a high affinity for a Notch receptoror a ligand of a Notch receptor. Certain fragments of fusion proteinsare protein fragments comprising at least part of the Notch bindingdomain of the polypeptide agent or antagonist fused to at least part ofa constant region of an immunoglobulin. The affinity is typically in therange of about 10⁻¹¹ to 10⁻¹² M, although the affinity can varyconsiderably with fragments of different sizes, ranging from 10⁻⁷ to10⁻¹³ M. In some embodiments, the fragment is about 10-110 amino acidsin length and comprises the Notch binding domain of the polypeptideagent or antagonist linked to at least part of a constant region of animmunoglobulin.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biological halflife or absorption of the protein. The moieties can also reduce oreliminate any undesirable side effects of the proteins and the like. Anoverview for those moieties can be found in Remington's PharmaceuticalSciences, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthesis methods, to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host.

In some embodiments of a recombinant method, a DNA sequence isconstructed by isolating or synthesizing a DNA sequence encoding awild-type protein of interest. Optionally, the sequence can bemutagenized by site-specific mutagenesis to provide functional analogsthereof. See, e.g. Zoeller et al., 1984, Proc. Nat Acad. Sci. USA81:5662-5066 and U.S. Pat. No. 4,588,585. Another method of constructinga DNA sequence encoding a polypeptide of interest would be by chemicalsynthesis using an oligonucleotide synthesizer. Such oligonucleotidescan be designed based on the amino acid sequence of the desiredpolypeptide and selecting those codons that are favored in the host cellin which the recombinant polypeptide of interest will be produced.

Standard methods can be applied to synthesize an isolated polynucleotidesequence encoding an isolated polypeptide of interest. For example, acomplete amino acid sequence can be used to construct a back-translatedgene. Further, a DNA oligomer containing a nucleotide sequence codingfor the particular isolated polypeptide can be synthesized. For example,several small oligonucleotides coding for portions of the desiredpolypeptide can be synthesized and then ligated. The individualoligonucleotides typically contain 5′ or 3′ overhangs for complementaryassembly.

Once assembled (by synthesis, site-directed mutagenesis, or anothermethod), the mutant DNA sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene is operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

Recombinant expression vectors may be used to amplify and express DNAencoding polypeptides. Recombinant expression vectors are replicable DNAconstructs which have synthetic or cDNA-derived DNA fragments encoding aNotch receptor fusion or a bioequivalent analog operatively linked tosuitable transcriptional or translational regulatory elements derivedfrom mammalian, microbial, viral or insect genes. A transcriptional unitgenerally comprises an assembly of (1) a genetic element or elementshaving a regulatory role in gene expression, for example,transcriptional promoters or enhancers, (2) a structural or codingsequence which is transcribed into mRNA and translated into protein, and(3) appropriate transcription and translation initiation and terminationsequences, as described in detail below. Such regulatory elements caninclude an operator sequence to control transcription. An origin ofreplication which usually confers the ability to replicate in a host anda selection gene to facilitate recognition of transformants canadditionally be incorporated. DNA regions are operatively linked whenthey are functionally related to each other. For example, DNA for asignal peptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Generally, “operativelylinked” means contiguous and, in the case of secretory leaders, meanscontiguous and in reading frame. Structural elements intended for use inyeast expression systems include a leader sequence enablingextracellular secretion of translated protein by a host cell.Alternatively, where recombinant protein is expressed without a leaderor transport sequence, it can include an N-terminal methionine residue.This residue can optionally be subsequently cleaved from the expressedrecombinant protein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirns. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Esherichia coli,including pCR1, pBR322, pMB9 and their derivatives, and wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a polypeptide include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or Bacilli. Higher eukatryoticcells include established cell lines of mammalian origin as describedherein. Cell-free translation systems could also be employed.Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described by Pouwels etal. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), therelevant disclosure of which is hereby incorporated by reference.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman 1981, Cell23:175, and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, 1988,Bio/Technology, 6:47.

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a cancer stem cell protein-Fc composition.Some or all of the foregoing purification steps, in variouscombinations, can also be employed to provide a homogeneous recombinantprotein.

Recombinant protein produced in bacterial culture is usually isolated byinitial extraction from cell pellets, followed by one or moreconcentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

In certain embodiments, the Notch-binding agent or antagonist comprisesan antibody. In certain embodiments, the antibody is isolated. Incertain embodiments, the antibody is substantially pure.

The present invention provides antibodies that compete for specificbinding to human Notch2 and/or Notch3 with an antibody comprising aheavy chain variable region comprising SEQ ID NO:14 and a light chainvariable region comprising SEQ ID NO:13. The present invention alsoprovides antibodies that compete for specific binding to human Notch2and/or Notch3 with an antibody that comprises, consists, or consistsessentially of a 59R1 IgG2 antibody comprising the heavy chain and lightchain of SEQ ID NOs:16 and 18 (with or without signal sequence),respectively, or as encoded by the DNA deposited with the ATCC on Oct.,15, 2008, and assigned designation number PTA-9547.

The invention further provides antibodies that specifically bind to oneor more Notch receptors, that comprise one, two, three, four, five,and/or six CDRs of SEQ ID NOs:5-10, 22-27, 30 or 51 with up to four(i.e., 0, 1, 2, 3, or 4) conservative amino acid substitutions (see,e.g., Table 1) per CDR. The invention also provides antibodies thatspecifically bind to one or more Notch receptors, that comprise one,two, three, four, five, and/or six CDRs of 59R1 (i.e., SEQ ID NOs:5-10),with up to four conservative amino acid substitutions per CDR. Thus, theinvention provides antibodies that specifically bind to one or morehuman Notch receptors that comprise one, two, three, four, five and/orsix of the CDRs of 59R1. In certain embodiments, the antibodies comprisethe heavy chain CDR3 of 59R1, with up to four conservative amino acidsubstitutions, and/or the light chain CDR3 of 59R1, with up to fourconservative amino acid substitutions. In some embodiments, the antibodycomprises (a) a heavy chain CDR1 comprising SSSGMS (SEQ ID NO:5), or avariant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; a heavy chain CDR2 comprising VIASSGSNTYYADSVKG (SEQ IDNO:6), or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions; and/or a heavy chain CDR3 comprising GIFFAI (SEQ IDNO:7), or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions; and/or (b) a light chain CDR1 comprisingRASQSVRSNYLA (SEQ ID NO:8), or a variant thereof comprising 1, 2, 3, or4 conservative amino acid substitutions; a light chain CDR2 comprisingGASSRAT (SEQ ID NO:9), or a variant thereof comprising 1, 2, 3, or 4conservative amino acid substitutions; and/or a light chain CDR3comprising QQYSNFPI (SEQ ID NO: 10), or a variant thereof comprising 1,2, 3, or 4 conservative amino acid substitutions.

The invention also provides antibodies that specifically bind to one ormore Notch receptors, that comprise one, two, three, four, five, and/orsix CDRs of 59R5 (i.e., SEQ ID NOs: 5, 6, 8-10, 51), with up to fourconservative amino acid substitutions per CDR. In certain embodiments,the antibodies comprise the heavy chain CDR3 of 59R5, with up to fourconservative amino acid substitutions, and/or the light chain CDR3 of59R5, with up to four conservative amino acid substitutions. In someembodiments, the antibody comprises (a) a heavy chain CDR1 comprisingSSSGMS (SEQ ID NO:5), or a variant thereof comprising 1, 2, 3, or 4conservative amino acid substitutions; a heavy chain CDR2 comprisingVIASSGSNTYYADSVKG (SEQ ID NO:6), or a variant thereof comprising 1, 2,3, or 4 conservative amino acid substitutions; and/or a heavy chain CDR3comprising SIFYTT (SEQ ID NO:51), or a variant thereof comprising 1, 2,3, or 4 conservative amino acid substitutions; and/or (b) a light chainCDR1 comprising RASQSVRSNYLA (SEQ ID NO:8), or a variant thereofcomprising 1, 2, 3, or 4 conservative amino acid substitutions; a lightchain CDR2 comprising GASSRAT (SEQ ID NO:9), or a variant thereofcomprising 1, 2, 3, or 4 conservative amino acid substitutions; and/or alight chain CDR3 comprising QQYSNFPI (SEQ ID NO: 10), or a variantthereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.In certain embodiments, the antibody comprises a heavy chain CDR1comprising SSSGMS (SEQ ID NO:5), a heavy chain CDR2 comprisingVIASSGSNTYYADSVKG (SEQ ID NO:6), and/or a heavy chain CDR3 comprisingSIFYTT (SEQ ID NO:51).

Also provided is an antibody that specifically binds human Notch2 and/orNotch3, wherein the antibody comprises a heavy chain CDR1 comprisingSSSGMS (SEQ ID NO:5), a heavy chain CDR2 comprising VIASSGSNTYYADSVKG(SEQ ID NO:6), and/or a heavy chain CDR-3 comprising(G/I)(I/S)F(F/Y)(A/P)(I/T/S/N) (SEQ ID NO:30). In certain embodiments,the heavy chain CDR3 is selected from the group consisting of SIFYPT(SEQ ID NO:22), SSFFAS (SEQ ID NO:23), SSFYAS (SEQ ID NO:24), SSFFAT(SEQ ID NO:25), SIFYPS (SEQ ID NO:26), and SSFFAN (SEQ ID NO:27). Incertain embodiments, the antibody comprises a heavy chain CDR1comprising SSSGMS (SEQ ID NO:5), a heavy chain CDR2 comprisingVIASSGSNTYYADSVKG (SEQ ID NO:6), and/or a heavy chain CDR3 comprisingGIFFAI (SEQ ID NO:7). In certain embodiments, the heavy chain CDR(s) arecontained within a variable region of an antibody heavy chain. Incertain embodiments, the antibody further comprises a light chain CDR1comprising RASQSVRSNYLA (SEQ ID NO:8), a light chain CDR2 comprisingGASSRAT (SEQ ID NO:9), and/or a light chain CDR3 comprising QQYSNFPI(SEQ TD NO:10). In certain embodiments, the light chain CDR(s) arecontained within a variable region of an antibody light chain. Incertain embodiments, the heavy chain CDR(s) and/or the light chainCDR(s) have been modified with 1, 2, 3, or 4 conservative amino acidsubstitutions. In certain embodiments, each of the CDR(s) have beenmodified by no more than 1-2 conservative amino acid substitutions.

For example, in certain embodiments, the invention provides an antibodythat specifically binds human Notch2 and/or Notch3, wherein the antibodycomprises: (a) a heavy chain CDR1 comprising SSSGMS (SEQ ID NO:5), aheavy chain CDR2 comprising VIASSGSNTYYADSVKG (SEQ ID NO:6), and a heavychain CDR3 comprising GIFFAI (SEQ ID NO:7); and/or (b) a light chainCDR1 comprising RASQSVRSNYLA (SEQ ID NO:8), a light chain CDR2comprising GASSRAT (SEQ ID NO:9), and a light chain CDR3 comprisingQQYSNFPI (SEQ ID NO:10). In some embodiments, the antibody comprisesboth the indicated light and heavy chain CDRs.

In some embodiments, the invention provides an antibody thatspecifically binds human Notch2 and/or Notch3, wherein the antibodycomprises: (a) a heavy chain CDR1 comprising SSSGMS (SEQ ID NO:5), aheavy chain CDR2 comprising VIASSGSNTYYADSVKG (SEQ ID NO:6), and a heavychain CDR3 comprising SIFYTT (SEQ ID NO:51); and/or (b) a light chainCDR1 comprising RASQSVRSNYLA (SEQ ID NO:8), a light chain CDR2comprising GASSRAT (SEQ ID NO:9), and a light chain CDR3 comprisingQQYSNFPI (SEQ ID NO:10). In certain embodiments, the antibody comprisesboth the indicated light and heavy chain CDRs.

The invention further provides an antibody that specifically binds humanNotch2 and/or Notch3, wherein the antibody comprises a light chain CDR Icomprising RASQSVRSNYLA (SEQ ID NO:8), a light chain CDR2 comprisingGASSRAT (SEQ ID NO:9), and/or a light chain CDR3 comprising QQYSNFPI(SEQ ID NO:10).

The invention also provides an antibody that specifically binds humanNotch2 and/or Notch3, wherein the antibody comprises: (a) a polypeptidehaving at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or at least about 98% sequence identity to SEQ ID NO:14or SEQ ID NO:20; and/or (b) a polypeptide having at least about 80%, atleast about 85%, at least about 90%, at least about 95%, or at leastabout 98% sequence identity to SEQ ID NO:13 or SEQ ID NO:19.Accordingly, in certain embodiments, the antibody comprises (a) a heavychain variable region having at least about 95% sequence identity to SEQID NO:14; and/or (b) a light chain variable region having at least about95% sequence identity to SEQ ID NO:13. In certain embodiments, theantibody comprises: (a) a polypeptide (e.g., a heavy chain variableregion) comprising SEQ ID NO:14 or SEQ ID NO:20; and/or (b) apolypeptide (e.g., a light chain variable region) comprising SEQ IDNO:13 or SEQ ID NO:19.

The invention also provides an antibody that specifically binds humanNotch2 and/or Notch3, wherein the antibody comprises: (a) a polypeptidehaving at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or at least about 98% sequence identity to SEQ IDNO:50; and/or (b) a polypeptide having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or at least about 98%sequence identity to SEQ ID NO:13. Accordingly, in certain embodiments,the antibody comprises (a) a heavy chain variable region having at leastabout 95% sequence identity to SEQ ID NO:50; and/or (b) a light chainvariable region having at least about 95% sequence identity to SEQ IDNO:13. In certain embodiments, the antibody comprises: (a) a polypeptide(e.g., a heavy chain variable region) comprising SEQ ID NO:50; and/or(b) a polypeptide (e.g., a light chain variable region) comprising SEQID NO:13.

In certain embodiments, the antagonists are antibodies that can mediatecomplement-dependent cytotoxicity or antibody-dependent cellularcytotoxicity to kill tumors expressing a target antigen. In certainalternative embodiments, the antibodies are directly conjugated totoxins or radioisotopes to mediate tumor cell killing. Furthermore,tumor survival depends on neo-vascularization, and in certainembodiments, the antibodies have an anti-angiogenic effect.

The present invention provides isolated antibodies against a Notchreceptor such as human Notch2 and/or Notch3. The antibody, or antibodyfragment, can be any monoclonal or polyclonal antibody that specificallyrecognizes the described Notch receptor. In some embodiments, thepresent invention provides monoclonal antibodies, or fragments thereof,that specifically bind to a Notch receptor described herein. In someembodiments, the monoclonal antibodies, or fragments thereof, arechimeric or humanized antibodies that specifically bind to theextracellular domain of a Notch receptor described herein. In otherembodiments, the monoclonal antibodies, or fragments thereof, are humanantibodies that specifically bind to the extracellular domain of a Notchreceptor described herein. In certain embodiments, the antibodies areIgG1 or IgG2 antibodies.

The antibodies against a Notch receptor find use in the experimental,diagnostic and therapeutic methods described herein. In certainembodiments, the antibodies of the present invention are used to detectthe expression of a Notch receptor in biological samples such as, forexample, a patient tissue biopsy, pleural effusion, or blood sample.Tissue biopsies can be sectioned and protein detected using, forexample, immunofluorescence or immunohistochemistry. Alternatively,individual cells from a sample are isolated, and protein expressiondetected on fixed or live cells by FACS analysis. Furthermore, theantibodies can be used on protein arrays to detect expression of a Notchreceptor, for example, on tumor cells, in cell lysates, or in otherprotein samples. In other embodiments, the antibodies of the presentinvention are used to inhibit the growth of tumor cells by contactingthe tumor cells with the antibodies either in in vitro cell based assaysor in vivo animal models. In still other embodiments, the antibodies areused to treat cancer in a human patient by administering atherapeutically effective amount of an antibody against a Notchreceptor.

Polyclonal antibodies can be prepared by any known method. Polyclonalantibodies are raised by immunizing an animal (e.g. a rabbit, rat,mouse, donkey, goat, etc.) by multiple subcutaneous or intraperitonealinjections of the relevant antigen (a purified peptide fragment,fill-length recombinant protein, fusion protein, etc.) optionallyconjugated to keyhole limpet hemocyanin (KLH), serum albumin, etc.diluted in sterile saline and combined with an adjuvant (e.g. Completeor incomplete Freund's Adjuvant) to form a stable emulsion. Thepolyclonal antibody is then recovered from blood, ascites and the like,of an animal so immunized. Collected blood is clotted, and the serumdecanted, clarified by centrifugation, and assayed for antibody titer.The polyclonal antibodies can be purified from serum or ascitesaccording to standard methods in the art including affinitychromatography, ion-exchange chromatography, gel electrophoresis,dialysis, etc.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, 1975, Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized as described above to elicit the production by lymphocytes ofantibodies that will specifically bind to an immunizing antigen.Alternatively, lymphocytes can be immunized in vitro. Followingimmunization, the lymphocytes are isolated and fused with a suitablemyeloma cell line using, for example, polyethylene glycol, to formhybridoma cells that can then be selected away from unfused lymphocytesand myeloma cells. Hybridomas that produce monoclonal antibodiesdirected specifically against a chosen antigen as determined byimmunoprecipitation, immunoblotting, or by an in vitro binding assaysuch as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay(ELISA) can then be propagated either in vitro culture using standardmethods (Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, 1986) or in vivo as ascites tumors in an animal. Themonoclonal antibodies can then be purified from the culture medium orascites fluid as described for polyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, express monoclonal antibodiesin the host cells. Also, recombinant monoclonal antibodies or fragmentsthereof of the desired species can be isolated from phage displaylibraries, e.g., as described herein.

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In otherembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Furthermore,site-directed or high-density mutagenesis of the variable region can beused to optimize specificity, affinity, etc. of a monoclonal antibody.

More generally, modified antibodies useful in the present invention maybe obtained or derived from any antibody. Further, the parent orprecursor antibody, or fragment thereof, used to generate the disclosedmodified antibodies may be murine, human, chimeric, humanized, non-humanprimate or primatized. In other embodiments the modified antibodies ofthe present invention can comprise single chain antibody constructs(such as that disclosed in U.S. Pat. No. 5,892,019, which isincorporated herein by reference) having altered constant domains asdescribed herein. Consequently, any of these types of antibodiesmodified in accordance with the teachings herein are compatible withthis invention.

According to the present invention, techniques can be adapted for theproduction of single-chain antibodies specific to a polypeptide of theinvention (see U.S. Pat. No. 4,946,778). In addition, methods can beadapted for the construction of Fab expression libraries (Huse et al.,1989, Science 246:1275-1281) to allow rapid and effective identificationof monoclonal Fab fragments with the desired specificity for Notch, orderivatives, fragments, analogs or homologs thereof. Antibody fragmentsthat contain the idiotypes to a polypeptide of the invention may beproduced by techniques in the art including, but not limited to: (a) anF(ab′)₂ fragment produced by pepsin digestion of an antibody molecule;(b) an Fab fragment generated by reducing the disulfide bridges of anF(ab′)₂ fragment, (c) an Fab fragment generated by the treatment of theantibody molecule with papain and a reducing agent, and (d) Fvfragments.

Bispecific antibodies are also within the scope of the invention.Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens (or, in certain embodiments, two different epitopes on the sameantigen). In the present case, one of the binding specificities is foran antigenic polypeptide of the invention (Notch, or a fragmentthereof), while the second binding target is any other antigen, andadvantageously is a cell surface protein, or receptor or receptorsubunit. Bispecific antibodies that comprise one antigen-binding sitethat specifically binds one human Notch receptor (e.g., Notch2) andfurther comprise a second, different antigen-binding site thatspecifically binds a second human Notch receptor (e.g., Notch3) areprovided.

Methods for making bispecific antibodies are known in the art.Traditionally the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain/light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, 1983, Nature 305:537-539). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatography.

Alternatively, in certain embodiments, the antibodies described hereinmay be monospecific. For example, in certain embodiments, each of theone or more antigen-binding sites that an antibody contains is capableof binding (or binds) the same one or more human Notch receptors (e.g.,Notch2, Notch3, or homologous epitopes on both Notch2 and Notch3). Incertain embodiments, an antigen-binding site of a monospecific antibodydescribed herein is capable of binding (or binds) both the EGF repeat 9of human Notch3 and EGF repeat 10 of Notch2.

Antibody variable domains with the desired binding specificities can befused to immunoglobulin constant domain sequences. The fusion is with animmunoglobulin heavy chain constant domain, comprising at least part ofthe hinge, CH2 and CH3 regions. The first heavy chain constant region(CH1) containing the site necessary for light chain binding can bepresent in at least one of the fusions. DNA encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host organism. Further details of generating bispecificantibodies can be found in Suresh et al., 1986, Methods in Enzymology121:210.

Bispecific antibodies can be prepared as full-length antibodies orantibody fragments. Techniques for generating bispecific antibodies fromantibody fragments have been described in the literature. For example,bispecific antibodies can be prepared using chemical linkage. Inaddition, Brennan et al., 1985, Science 229:81 describe a procedurewherein intact antibodies are proteolytically cleaved to generateF(ab′)₂ fragments.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies (Shalaby et al., 1992,J. Exp. Med. 175:217-225). These methods can be used in the productionof a fully humanized bispecific antibody F(ab′)₂ molecule.

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared (Tutt et al., 1991, J.Immunol. 147:60).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in a polypeptide of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG so as to focus cellular defense mechanismsto the cell expressing the particular antigen. Bispecific antibodies canalso be used to direct cytotoxic agents to cells which express aparticular antigen. These antibodies possess an antigen-binding arm andan arm which binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA.

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It iscontemplated that the antibodies can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins can be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For the purposes of the present invention, it should be appreciated thatmodified antibodies can comprise any type of variable region thatprovides for the association of the antibody with the polypeptides ofNotch. In this regard, the variable region may comprise or be derivedfrom any type of mammal that can be induced to mount a humoral responseand generate immunoglobulins against the desired tumor associatedantigen. As such, the variable region of the modified antibodies can be,for example, of human, murine, non-human primate (e.g. cynomolgusmonkeys, macaques, etc.) or lupine origin. In some embodiments both thevariable and constant regions of the modified immunoglobulins are human.In other embodiments the variable regions of compatible antibodies(usually derived from a non-human source) can be engineered orspecifically tailored to improve the binding properties or reduce theimmunogenicity of the molecule. In this respect, variable regions usefulin the present invention can be humanized or otherwise altered throughthe inclusion of imported amino acid sequences.

In some embodiments, of the present invention the monoclonal antibodyagainst a Notch receptor is a humanized antibody. Humanized antibodiesare antibodies that contain minimal sequences from non-human (e.g.,murine) antibodies within the variable regions. Such antibodies are usedtherapeutically to reduce antigenicity and HAMA (human anti-mouseantibody) responses when administered to a human subject. In practice,humanized antibodies are typically human antibodies with minimum to nonon-human sequences. A human antibody is an antibody produced by a humanor an antibody having an amino acid sequence corresponding to anantibody produced by a human.

Humanized antibodies can be produced using various techniques known inthe art. An antibody can be humanized by substituting the CDR of a humanantibody with that of a non-human antibody (e.g., mouse, rat, rabbit,hamster, etc.) having the desired specificity, affinity, and/orcapability (Jones et al., 1986, Nature 321:522-525; Riechmann et al.,1988, Nature 332:323-327; Verhoeyen et al., 1988, Science239:1534-1536). The humanized antibody can be further modified by thesubstitution of additional residues either in the Fv framework regionand/or within the replaced non-human residues to refine and optimizeantibody specificity, affinity, and/or capability.

As an alternative to humanization, human antibodies can be generated.Human antibodies can be prepared using various techniques known in theart, including from transgenic animals, phage libraries, and in vitroactivated human B cells.

For example, it is now possible to produce transgenic animals (e.g.,mice) containing human immunoglobulin loci that are capable, uponimmunization, of producing a full repertoire of human antibodies in theabsence of endogenous immunoglobulin production. For example, it hasbeen described that the homozygous deletion of the antibody heavy-chainjoining region (J) gene in chimeric and germ-line mutant mice results incomplete inhibition of endogenous antibody production. Transfer of thehuman germ-line immunoglobulin gene array into such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. See, e.g., Jakobovits et al., 1993, Proc. Nat. Acad. Sci. USA90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggemann et al.,1993, Year in Immuno. 7:33; U.S. Pat. Nos. 5,545,806; 5,569,825;5,591,669; 5,545,807; 5,545,807; 5,625,126; 5,633,425; and 5,661,016;and WO 97/17852.

Alternatively, phage display technology can be used to produce humanantibodies and antibody fragments in vitro, from immunoglobulin variable(V) domain gene repertoires from unimmunized donors. According to thistechnique, antibody V domain genes are cloned in-frame into either amajor or minor coat protein gene of a filamentous bacteriophage, such asM13 or fd, and displayed as functional antibody fragments on the surfaceof the phage particle. Because the filamentous particle contains asingle-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. Thus, the phagemimics some of the properties of the B-cell. Phage display can beperformed in a variety of formats. Several sources of V-gene segmentscan be used for phage display. A diverse array of anti-oxazoloneantibodies have been isolated from a small random combinatorial libraryof V genes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolated.Methods of selecting human antibodies from a phage library, where thatphage library expresses human antibodies are well known in the art(Vaughan et al., 1996, Nature Biotechnology 14:309-314; Sheets et al.,1998, PNAS 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol.227:381; McCafferty et al., 1990, Nature 348:552-554; Clackson et al.,1991, Nature 352:624-628; and Marks et al., 1991, J. Mol. Biol.,222:581-597). Techniques for the generation and use of antibody phagelibraries are also described in U.S. Pat. Nos. 5,969,108; 6,172,197;5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081;6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., 2008,J. Mol. Bio. 376:1182-1200 (each of which is incorporated by referencein its entirety). Affinity maturation strategies, such as chainshuffling (Marks et al., 1992, Bio/Technology 10:779-783, incorporatedby reference in its entirety), are known in the art and may be employedto generate high affinity human antibodies.

Human antibodies can also be directly prepared using various techniquesknown in the art. Immortalized human B lymphocytes immunized in vitro orisolated from an immunized individual that produce an antibody directedagainst a target antigen can be generated. (See, for example, Cole etal., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77; Boerner et al., 1991, J. Immunol., 147 (1):86-95; U.S. Pat. Nos.5,750,373; 5,567,610; and 5,229,275).

It will be appreciated that grafting the entire non-human variabledomains onto human constant regions will produce “classic” chimericantibodies. In the context of the present application the term “chimericantibodies” will be held to mean any antibody wherein the immunoreactiveregion or site is obtained or derived from a first species and theconstant region (which may be intact, partial or modified in accordancewith this invention) is obtained from a second species. In someembodiments, the antigen binding region or site will be from a non-humansource (e.g., mouse) and the constant region is human. While theimmunogenic specificity of the variable region is not generally affectedby its source, a human constant region is less likely to elicit animmune response from a human subject than would the constant region froma non-human source.

The variable domains in both the heavy and light chains are altered byat least partial replacement of one or more CDRs and, if necessary, bypartial framework region replacement and sequence modification. Althoughthe CDRs may be derived from an antibody of the same class or evensubclass as the antibody from which the framework regions are derived,it is envisaged that the CDRs will be derived from an antibody ofdifferent class and preferably from an antibody from a differentspecies. It must be emphasized that it may not be necessary to replaceall of the CDRs with the complete CDRs from the donor variable region totransfer the antigen binding capacity of one variable domain to another.Rather, it may only be necessary to transfer those residues that arenecessary to maintain the activity of the antigen binding site. Giventhe explanations set forth in U.S. Pat. Nos. 5,585,089; 5,693,761; and5,693,762, it will be well within the art, either by carrying outroutine experimentation or by trial and error testing to obtain afunctional antibody with reduced immunogenicity.

Alterations to the variable region notwithstanding, it will beappreciated that the modified antibodies of this invention will compriseantibodies, or immunoreactive fragments thereof, in which at least afraction of one or more of the constant region domains has been deletedor otherwise altered so as to provide desired biochemical and/orbiological characteristics such as increased tumor localization orreduced serum half-life when compared with an antibody of approximatelythe same immunogenicity comprising a native or unaltered constantregion. In some embodiments, the constant region of the modifiedantibodies will comprise a human constant region. Modifications to theconstant region compatible with this invention comprise additions,deletions or substitutions of one or more amino acids in one or moredomains. That is, the modified antibodies disclosed herein may comprisealterations or modifications to one or more of the three heavy chainconstant domains (CH1, CH2 or CH3) and/or to the light chain constantdomain (CL). In some embodiments of the invention, modified constantregions wherein one or more domains are partially or entirely deletedare contemplated. In other embodiments, the modified antibodies willcomprise domain deleted constructs or variants wherein the entire CH2domain has been removed (ΔCH2 constructs). In still other embodiments,the omitted constant region domain will be replaced by a short aminoacid spacer (e.g., 10 residues) that provides some of the molecularflexibility typically imparted by the absent constant region.

Besides their configuration, it is known in the art that the constantregion mediates several effector functions. For example, binding of theC1 component of complement to antibodies activates the complementsystem. Activation of complement is important in the opsonization andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and can also be involved in autoimmunehypersensitivity. Further, antibodies bind to cells via the Fc region,with a Fc receptor site on the antibody Fc region binding to a Fcreceptor (FcR) on a cell. There are a number of Fc receptors which arespecific for different classes of antibody, including IgG (gammareceptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mureceptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production. Although various Fc receptors and receptorsites have been studied to a certain extent, there is still much whichis unknown about their location, structure and functioning.

While not limiting the scope of the present invention, it is believedthat antibodies comprising constant regions modified as described hereinprovide for altered effector functions that, in turn, affect thebiological profile of the administered antibody. For example, thedeletion or inactivation (through point mutations or other means) of aconstant region domain may reduce Fc receptor binding of the circulatingmodified antibody thereby increasing tumor localization. In other casesit may be that constant region modifications, consistent with thisinvention, moderate complement binding and thus reduce the serum halflife and nonspecific association of a conjugated cytotoxin. Yet othermodifications of the constant region may be used to eliminate disulfidelinkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. Similarly, modifications to the constant region inaccordance with this invention may easily be made using well knownbiochemical or molecular engineering techniques.

It will be noted that the modified antibodies may be engineered to fusethe CH3 domain directly to the hinge region of the respective modifiedantibodies. In other constructs it may be desirable to provide a peptidespacer between the hinge region and the modified CH2 and/or CH3 domains.For example, compatible constructs could be expressed wherein the CH2domain has been deleted and the remaining CH3 domain (modified orunmodified) is joined to the hinge region with a 5-20 amino acid spacer.Such a spacer may be added, for instance, to ensure that the regulatoryelements of the constant domain remain free and accessible or that thehinge region remains flexible. However, it should be noted that aminoacid spacers may, in some cases, prove to be immunogenic and elicit anunwanted immune response against the construct. Accordingly, any spaceradded to the construct should be relatively non-immunogenic or, evenomitted altogether if the desired biochemical and/or biologicalqualities of the modified antibodies are to be maintained.

Besides the deletion of whole constant region domains, it will beappreciated that the antibodies of the present invention may be providedby the partial deletion or substitution of a few or even a single aminoacid. For example, the mutation of a single amino acid in selected areasof the CH2 domain may be enough to substantially reduce Fc binding andthereby increase tumor localization. Similarly, it may be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g., complement Clq binding) to bemodulated. Such partial deletions of the constant regions may improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies may be modified through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g., Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Yet other embodiments may comprise the additionof one or more amino acids to the constant region to enhance desirablecharacteristics such as effector function or provide for more cytotoxinor carbohydrate attachment. In such embodiments it can be desirable toinsert or replicate specific sequences derived from selected constantregion domains.

This invention also encompasses bispecific antibodies that specificallyrecognize a Notch receptor. Bispecific antibodies are antibodies thatare capable of specifically recognizing and binding at least twodifferent epitopes. The different epitopes can either be within the samemolecule (e.g., the same Notch receptor polypeptide) or on differentmolecules. For example, the antibodies can specifically recognize andbind a Notch receptor as well as, for example, 1) an effector moleculeon a leukocyte such as a T-cell receptor (e.g., CD3) or Fc receptor(e.g., CD64, CD32, or CD16) or 2) a cytotoxic agent as described indetail herein. Bispecific antibodies can be intact antibodies orantibody fragments. Techniques for making bispecific antibodies arecommon in the art (Millstein et al., 1983, Nature 305:537-539; Brennanet al., 1985, Science 229:81; Suresh et al, 1986, Methods in Enzymol.121:120; Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalaby et al.,1992, J. Exp. Med. 175:217-225; Kostelny et al., 1992, J. Immunol. 148:1547-1553; Gruber et al., 1994, J. Immunol. 152:5368; and U.S. Pat. No.5,731,168).

In certain embodiments of the invention, it can be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. Various techniques are known for theproduction of antibody fragments. Traditionally, these fragments arederived via proteolytic digestion of intact antibodies (for exampleMorimoto et al., 1993, Journal of Biochemical and Biophysical Methods24:107-117 and Brennan et al., 1985, Science, 229:81). However, thesefragments are now typically produced directly by recombinant host cellsas described herein. Thus Fab, Fv, and scFv antibody fragments can allbe expressed in and secreted from E. coli or other host cells, thusallowing the production of large amounts of these fragments.Alternatively, such antibody fragments can be isolated from the antibodyphage libraries discussed herein. The antibody fragments can also belinear antibodies as described in U.S. Pat. No. 5,641,870, for example,and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent.

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent. Cytotoxic agents includechemotherapeutic agents, growth inhibitory agents, toxins (e.g., anenzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), radioactive isotopes (i.e., aradioconjugate), etc. Chemotherapeutic agents useful in the generationof such immunoconjugates include, for example, methotrexate, adriamicin,doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents. Enzymatically active toxins and fragments thereofthat can be used include diphtheria. A chain, nonbinding activefragments of diphtheria, toxin, exotoxin A chain, ricin A chain, abrin Achain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, andPAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. Conjugates of the antibody andcytotoxic agent are made using a variety of bifunctionalprotein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azidocompounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Conjugatesof an antibody and one or more small molecule toxins, such as acalicheamicin, maytansinoids, a trichothene, and CC1065, and thederivatives of these toxins that have toxin activity, can also be used.

Conjugate antibodies are composed of two covalently joined antibodies.Such antibodies have, for example, been proposed to target immune cellsto unwanted cells (U.S. Pat. No. 4,676,980). It is contemplated that theantibodies can be prepared in vitro using known methods in syntheticprotein chemistry, including those involving crosslinking agents. Forexample, immunotoxins can be constructed using a disulfide exchangereaction or by forming a thioether bond. Examples of suitable reagentsfor this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

In some embodiments, the antibody of the invention contains human Fcregions that are modified to enhance effector function, for example,antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complementdependent cytotoxicity (CDC). This can be achieved by introducing one ormore amino acid substitutions in an Fc region of the antibody. Forexample, cysteine residue(s) can be introduced in the Fc region to allowinterchain disulfide bond formation in this region to improvecomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC) (Caron et al., 1992, J. Exp Med. 176:1191-1195;Shopes, 1992, Immunol. 148:2918-2922). Homodimeric antibodies withenhanced anti-tumor activity can also be prepared usingheterobifunctional cross-linkers as described in Wolff et al., 1993,Cancer Research 53:2560-2565. Alternatively, an antibody can beengineered which has dual Fc regions (Stevenson et al., 1989,Anti-Cancer Drug Design 3:219-230).

Regardless of how useful quantities are obtained, the antibodies of thepresent invention can be used in any one of a number of conjugated (i.e.an immunoconjugate) or unconjugated forms. The antibodies of thisinvention can be used in a nonconjugated or “naked” form to harness thesubject's natural defense mechanisms including complement-dependentcytotoxicity (CDC) and antibody dependent cellular toxicity (ADCC) toeliminate the malignant cells. In some embodiments, the antibodies canbe conjugated to radioisotopes, including, but not limited to, ⁹⁰Y,¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹³¹In, ²¹²Bi, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho,¹⁷⁷Lu, ¹⁸⁶Re and ¹⁸⁸Re using any one of a number of well known chelatorsor direct labeling. In other embodiments, the disclosed compositions cancomprise antibodies coupled to drugs, prodrugs or biological responsemodifiers such as methotrexate, adriamycin, and lymphokines such asinterferon. Still other embodiments of the present invention comprisethe use of antibodies conjugated to specific biotoxins such as ricin ordiptheria toxin. In yet other embodiments the modified antibodies can becomplexed with other immunologically active ligands (e.g., antibodies orfragments thereof) wherein the resulting molecule binds to both theneoplastic cell and an effector cell such as a T cell. The selection ofwhich conjugated or unconjugated modified antibody to use will depend ofthe type and stage of cancer, use of adjunct treatment (e.g.,chemotherapy or external radiation) and patient condition. It will beappreciated that one could readily make such a selection in view of theteachings herein.

The preparation and characterization of anti-Notch antibodies is alsotaught, e.g., in U.S. Patent Application Publication No. 2008/0131434,which is incorporated by reference herein in its entirety.

In certain embodiments, the Notch-binding agent or antagonist is apolypeptide that is not an antibody. A variety of methods foridentifying and producing non-antibody polypeptides that bind with highaffinity to a protein target are known in the art. See, e.g., Skerra,2007, Curr. Opin. Biotechnol., 18:295-304; Hosse et al., 2006, ProteinScience, 15:14-27; Gill et al., 2006, Curr. Opin. Biotechnol.,17:653-658; Nygren, 2008, FEBS J., 275:2668-76; and Skerra, 2008, FEBSJ., 275:2677-83, each of which is incorporated by reference herein inits entirety. In certain embodiments, phage display technology has beenused to identify/produce the Notch-binding polypeptide. In certainembodiments, the polypeptide comprises a protein scaffold of a typeselected from the group consisting of protein A, a lipocalin, afibronectin domain, an ankyrin consensus repeat domain, and thioredoxin.

In some embodiments, the agent is a non-protein molecule. In certainembodiments, the agent is a small molecule. Combinatorial chemistrylibraries and techniques useful in the identification of non-proteinNotch-binding agents are known to those skilled in the art. See, e.g.,Kennedy et al., 2008, J. Comb. Chem., 10:345-354; Dolle et al, 2007, J.Comb. Chem., 9:855-902; and Bhattacharyya, 2001, Curr. Med. Chem.,8:1383-404, each of which is incorporated by reference herein in itsentirety. In certain further embodiments, the agent is a carbohydrate, aglycosaminoglycan, a glycoprotein, or a proteoglycan.

In certain embodiments, the agent is a nucleic acid aptamer. Aptamersare polynucleotide molecules that have been selected (e.g., from randomor mutagenized pools) on the basis of their ability to bind to anothermolecule. In some embodiments, the aptamer comprises a DNApolynucleotide. In certain alternative embodiments, the aptamercomprises an RNA polynucleotide. In certain embodiments, the aptamercomprises one or more modified nucleic acid residues. Methods ofgenerating and screening nucleic acid aptamers for binding to proteinsare well known in the art. See, e.g., U.S. Pat. Nos. 5,270,163;5,683,867; 5,763,595; 6,344,321; 7,368,236; 5,582,981; 5,756,291;5,840,867; 7,312,325; and 7,329,742; International Patent PublicationNos. WO 02/077262; WO 03/070984; U.S. Patent Application PublicationNos. 2005/0239134; 2005/0124565; and 2008/0227735, each of which isincorporated by reference herein in its entirety.

The antibodies of the present invention can be assayed forimmunospecific binding by any method known in the art. The immunoassayswhich can be used include, but are not limited to, competitive andnon-competitive assay systems using techniques such as Biacore analysis,FACS analysis, immunofluorescence, immunocytochemistry, Western blotanalysis, radioimmunoassay, ELISA, “sandwich” immunoassay,immunoprecipitation assay, precipitin reaction, gel diffusion precipitinreaction, immunodiffusion assay, agglutination assay,complement-fixation assay, immunoradiometric assay, fluorescentimmunoassay, and protein A immunoassay. Such assays are routine and wellknown in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocolsin Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, whichis incorporated by reference herein in its entirety).

In some embodiments, of the present invention the immunospecificity ofan antibody against a Notch receptor is determined using ELISA. An ELISAassay comprises preparing antigen, coating wells of a 96 well microtiterplate with antigen, adding the antibody against a Notch receptorconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well,incubating for a period of time and detecting the presence of theantigen. Alternatively the antibody against a Notch receptor is notconjugated to a detectable compound, but instead a second conjugatedantibody that recognizes the antibody against a Notch receptor is addedto the well. Further, instead of coating the well with the antigen, theantibody against a Notch receptor can be coated to the well and a secondantibody conjugated to a detectable compound can be added following theaddition of the antigen to the coated well. The parameters that can bemodified to increase the signal detected, as well as other variations ofELISAs are well known in the art (see e.g. Ausubel et al., eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York at 11.2.1).

The binding affinity of an antibody to a Notch receptor and the off-rateof an antibody-antigen interaction can be determined by competitivebinding assays. One example of a competitive binding assay is aradioimmunoassay comprising the incubation of labeled antigen (e.g., ³Hor ¹²⁵I), or fragment or variant thereof, with the antibody of interestin the presence of increasing amounts of unlabeled antigen followed bythe detection of the antibody bound to the labeled antigen. The affinityof the antibody against a Notch receptor and the binding off-rates canbe determined from the data by Scatchard plot analysis. In someembodiments, Biacore kinetic analysis is used to determine the bindingon and off rates of antibodies against a Notch receptor. Biacore kineticanalysis comprises analyzing the binding and dissociation of antibodiesfrom chips with immobilized Notch antigens on their surface.

The invention provides isolated polynucleotides encoding thepolypeptides of SEQ ID NOs:2, 4, 13, 14, 16, 18, 19, 20, 39, 40, 49, 50,52, 53, 54, 55, 56, or 57 as well as the polynucleotides of SEQ ID NOs:1, 3, 15, 17, 47 or 48. The polynucleotides of the invention can be inthe form of RNA or in the form of DNA, wherein DNA includes cDNA,genomic DNA, and synthetic DNA. The DNA can be double-stranded orsingle-stranded, and if single-stranded it can be the coding strand ornon-coding (anti-sense) strand. Thus, the term “polynucleotide encodinga polypeptide” encompasses a polynucleotide which includes only codingsequences for the polypeptide as well as a polynucleotide which includesadditional coding and/or non-coding sequences. In some embodiments, theinvention provides a polynucleotide that hybridizes to a polynucleotideencoding the polypeptides of SEQ ID NOs:2, 4, 13, 14, 16, 18, 19, 20,39, 40, 49, 50, 52, 53, 54, 55, 56, or 57. In some embodiments, thepolynucleotides hybridize to the polynucleotides of SEQ ID NOs: 1, 3,15, 17, 47, 48, 58, 59 or 60. In some embodiments, the polynucleotideshybridize under stringent hybridization conditions.

As used herein, the phrases “hybridizes” or “selectively hybridizes” or“specifically hybridizes” refer to the binding or duplexing of amolecule only to a particular nucleotide sequence under stringenthybridization conditions when that sequence is present in a complexmixture (e.g., a library of DNAs or RNAs). See, e.g., Andersen (1998)Nucleic Acid Hybridization Springer-Verlag; Ross (ed. 1997) Nucleic AcidHybridization Wiley.

As used herein, the phrase “stringent hybridization conditions” refersto conditions under which a probe or other polynucleotide will hybridizeto its target subsequence or other complementary sequence, typically ina complex mixture of nucleic acid, but generally to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength. The Tm is the temperature (under defined ionicstrength, pH, and nucleic concentration) at which 50% of the probescomplementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionscan also be achieved with the addition of destabilizing agents such asformamide. For high stringency hybridization, a positive signal is atleast two times background, or 10 times background hybridization.Exemplary high stringency or stringent hybridization conditions include:50% formamide, 5×SSC, and 1% SDS incubated at 42° C. or 5×SSC and 1% SDSincubated at 65° C., with a wash in 0.2×SSC and 0.1% SDS at 65° C. ForPCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures can vary from about 32°C. to about 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures cart range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C. to 95° C. for 30-120 see, an annealingphase lasting 30-120 sec, and an extension phase of about 72° C. for 1-2min.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs, andderivatives. The variant of the polynucleotide can be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

As hereinabove indicated, the polynucleotide can have a coding sequencewhich is a naturally occurring allelic variant of the coding sequence ofthe disclosed polypeptides. As known in the art, an allelic variant isan alternate form of a polynucleotide sequence which has a substitution,deletion or addition of one or more nucleotides that does notsubstantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide can be fused in the same readingframe to a polynucleotide which aids in expression and secretion of apolypeptide from a host cell, for example, a leader sequence whichfunctions as a secretory sequence for controlling transport of apolypeptide from the cell. The polypeptide having a leader sequence is apreprotein and can have the leader sequence cleaved by the host cell toform the mature form of the polypeptide. The polynucleotides can alsoencode for a proprotein which is the mature protein plus additional 5′amino acid residues. A mature protein having a prosequence is aproprotein and is an inactive form of the protein. Once the prosequenceis cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention canencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and presequence (leadersequence).

The polynucleotides of the present invention can also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. For example,the marker sequence can be a hexa-histidine tag supplied by a pQE-9vector to provide for purification of the mature polypeptide fused tothe marker in the case of a bacterial host. Or for example, the markersequence can be a hemagglutinin (HA) tag when a mammalian host, e.g.COS-7 cells, is used. The HA tag corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767).

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 90% identical, 95% identical, and in some embodiments, at least96%, 97%, 98% or 99% identical to SEQ ID NOs:1, 3, 15, 17, 47, 48, 58,59 or 60. In some embodiments, the polynucleotides comprising anucleotide sequence at least 90% identical, 95% identical, and in someembodiments, at least 96%, 97%, 98% or 99% identical hybridize to thepolynucleotides of SEQ ID NOs:1, 3, 15, 17, 47, 48, 58, 59 or 60. Insome embodiments, the polynucleotides comprising a nucleotide sequencesat least 90% identical, 95% identical, and in some embodiments, at least96%, 97%, 98% or 99% identical hybridize to the polynucleotides of SEQID NOs:58, 59 or 60. In some embodiments, the polynucleotides hybridizeunder stringent hybridization conditions. In some embodiments, thepolynucleotides hybridize to the polynucleotides of SEQ ID NO:58, 59 or60 under stringent hybridization conditions. By a polynucleotide havinga nucleotide sequence at least, for example, 95% “identical” to areference nucleotide sequence is intended that the nucleotide sequenceof the polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence can include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence can be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence can be inserted into the referencesequence. These mutations of the reference sequence can occur at theamino- or carboxy-terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among nucleotides in the reference sequence or inone or more contiguous groups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule hasa certainty percent sequence identity to a reference sequence (forexample, has at least about 80%, at least about 90%, at least about 95%,or at least about 97% sequence identity to a reference sequence or is95%, 96%, 97%, 98% or 99% identical to the reference sequence) can bedetermined conventionally using known computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). Bestfit uses the local homology algorithmof Smith and Waterman, 1981, Advances in Applied Mathematics 2: 482-489,to find the best segment of homology between two sequences. When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference nucleotide sequence and that gaps in homology ofup to 5% of the total number of nucleotides in the reference sequenceare allowed.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some embodiments,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host such as changing codons in the human mRNA to thosepreferred by a bacterial host such as E. coli.

The present invention further provides pharmaceutical compositionscomprising antagonists (e.g., antibodies) that target a Notch receptor.These pharmaceutical compositions find use in inhibiting tumor cellgrowth and treating cancer in human patients.

Formulations are prepared for storage and use by combining a purifiedNotch-binding agent or antagonist (e.g., antibody) of the presentinvention with a pharmaceutically acceptable carrier, excipient, and/orstabilizer as a sterile lyophilized powder, aqueous solution, etc.(Remington, The Science and Practice of Pharmacy 20th Edition MackPublishing, 2000). Suitable carriers, excipients, or stabilizerscomprise: nontoxic buffers such as phosphate, citrate, and other organicacids; salts such as sodium chloride; antioxidants such as ascorbic acidand methionine; preservatives such as octadecyldimethylbenzyl ammoniumchloride, hexamethonium chloride, benzalkonium chloride, benzethoniumchloride, phenol, butyl or benzyl alcohol, alkyl parabens, such asmethyl or propyl paraben, catechol, resorcinol, cyclohexanol,3-pentanol, and m-cresol; low molecular weight polypeptides (less thanabout 10 amino acid residues); proteins such as serum albumin, gelatin,and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,and lysine; carbohydrates such as monosacchandes, disaccharides,glucose, mannose, and dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose and sorbitol; salt-formingcounter-ions such as sodium; metal complexes such as Zn-proteincomplexes; and/or non-ionic surfactants such as TWEEN and polyethyleneglycol (PEG).

The pharmaceutical composition of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical (such as to mucous membranesincluding vaginal and rectal delivery) using transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders; pulmonary (e.g., by inhalation or insufflation of powdersor aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal); oral; or parenteral including intravenous,intraarterial, subcutaneous, intraperitoneal, intratumoral, orintramuscular injection or infusion; or intracranial (e.g., intrathecalor intraventricular) administration.

The therapeutic formulation can be in unit dosage form. Suchformulations include tablets, pills, capsules, powders, granules,solutions or suspensions in water or non-aqueous media, or suppositoriesfor oral, parenteral, or rectal administration or for administration byinhalation. In solid compositions such as tablets the principal activeingredient is mixed with a pharmaceutical carrier. Conventionaltableting ingredients include corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother diluents (e.g., water) to form a solid preformulation compositioncontaining a homogeneous mixture of a compound of the present invention,or a non-toxic pharmaceutically acceptable salt thereof. The solidpreformulation composition is then subdivided into unit dosage forms ofthe type described above. The tablets, pills, etc. of the novelcomposition can be coated or otherwise compounded to provide a dosageform affording the advantage of prolonged action. For example, thetablet or pill can comprise an inner composition covered by an outercomponent. Furthermore, the two components can be separated by anenteric layer that serves to resist disintegration and permits the innercomponent to pass intact through the stomach or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

Pharmaceutical formulations include antagonists (e.g., antibodies) ofthe present invention complexed with liposomes (Epstein, et al., 1985,Proc. Natl. Acad. Sci. USA 82:3688; Hwang, et al., 1980, Proc. Natl.Acad. Sci. USA 77:4030; and U.S. Pat. Nos. 4,485,045 and 4,544,545).Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556. Some liposomes can be generated by the reverse phaseevaporation with a lipid composition comprising phosphatidylcholine,cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).Liposomes are extruded through filters of defined pore size to yieldliposomes with the desired diameter.

The antagonist (e.g., antibody) can also be entrapped in microcapsules.Such microcapsules are prepared, for example, by coacervation techniquesor by interfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nanoparticles andnanocapsules) or in macroemulsions as described in Remington's, TheScience and Practice of Pharmacy, 20th Ed., Mack Publishing (2000).

In addition sustained-release preparations can be prepared. Suitableexamples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles (e.g., films ormicrocapsules). Examples of sustained-release matrices includepolyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) orpoly(v nylalcohol), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and 7 ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the Lupron Depot (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), sucrose acetateisobutyrate, and poly-D(−)-3-hydroxybutyric acid.

In certain embodiments, the pharmaceutical compositions comprise boththe Notch-binding agent or antagonist and a second therapeutic agent. Incertain embodiments, the second therapeutic agent is an anti-canceragent and/or an anti-angiogenic agent.

The present invention provides methods for inhibiting the growth orproliferation of tumorigenic cells expressing a Notch receptor using theNotch receptor antagonists described herein. In some embodiments, themethods comprise inhibiting the growth of tumorigenic cells expressing aNotch2 and/or Notch3 receptor using any one the antibodies orpolypeptides described herein. In some embodiments, the method ofinhibiting the growth of tumorigenic cells expressing a Notch receptorcomprises contacting the cell with an antagonist against a Notchreceptor in vitro. For example, an immortalized cell line or a cancercell line that expresses a Notch receptor is cultured in medium to whichis added an antibody which specifically binds to Notch2 and/or Notch3and inhibits cell growth. Or tumor cells and/or tumor stem cells areisolated from a patient sample such as, for example, a tissue biopsy,pleural effusion, or blood sample and cultured in medium to which isadded an antibody which specifically binds to Notch2 and/or Notch3 andinhibits cell growth. In some embodiments, the antagonist is an antibodythat specifically recognizes an epitope of a Notch2 and/or Notch3receptor.

In some embodiments, the method of inhibiting the growth orproliferation of tumorigenic cells expressing a Notch receptor comprisescontacting the cell with an antagonist against a Notch receptor (e.g.,an antagonist of Notch2 and/or Notch3) in vivo. In certain embodiments,contacting a tumorigenic cell with an antagonist to a Notch receptor isundertaken in an animal model. For example, xenografts expressing aNotch receptor are grown in immunocompromised mice (e.g., NOD/SCIDmice). The mice are administered an antagonist to the Notch receptor toinhibit tumor growth. Alternatively, cancer stem cells that express aNotch receptor are isolated from a patient sample such as, for example,a tissue biopsy, pleural effusion, or blood sample and injected intoimmunocompromised mice. The mice are then administered an antagonistagainst the Notch receptor to inhibit tumor cell growth. In someembodiments, the antagonist of a Notch receptor is administered at thesame time or shortly after introduction of tumorigenic cells into theanimal to prevent tumor growth. In other embodiments, the antagonist ofa Notch receptor is administered as a therapeutic after the tumorigeniccells have grown to a specified size. In some embodiments, theantagonist is a Notch receptor protein fusion that specifically binds toa Notch receptor. In certain embodiments, the antagonist is an antibodythat specifically recognizes an epitope of a Notch receptor. In someembodiments, the antibody is any one of the antibodies or polypeptidesdescribed herein.

In certain embodiments, contacting a tumorigenic cell with an antagonistto a Notch receptor is undertaken in a human patient diagnosed withcancer. In some embodiments, the antagonist is an antibody thatspecifically binds to a Notch receptor. In other embodiments, theantagonist is an antibody that specifically recognizes an epitope of aNotch receptor. For example, the invention provides method of inhibitinggrowth of a tumor in a subject, comprising administering to the subjecta therapeutically effective amount of an antagonist of human Notch2and/or Notch3. In some embodiments, the antagonist is an antibody thatbinds to Notch2. In some embodiments, the antagonist is an antibody thatbinds to Notch3. In some embodiments, the antagonist is an antibody thatbinds to Notch2 and Notch3. In some embodiments, the antagonist is anantibody or polypeptide as described in any one of the aforementionedaspects or embodiments, as well as any other aspects or embodimentsdescribed herein. In certain embodiments, the tumor comprises aninactivating deletion or mutation in the phosphatase and tensin homolog(PTEN) gene.

The invention further provides methods of inhibiting Notch signaling(e.g., Notch2 and/or Notch3) in a cell, comprising contacting the cellwith an effective amount of the Notch antagonist. These methods may bein vivo or in vitro. In some embodiments, the Notch antagonist is anantibody. In some embodiments, the methods comprise inhibiting Notch2signaling in a cell comprising contacting the cell with an effectiveamount of any one of the antibodies or polypeptides of theaforementioned aspects or embodiments, as well as any other aspects orembodiments described herein. In some embodiments, the Notch antagonistis an antibody. In some embodiments, the methods comprise inhibitingNotch3 signaling in a cell comprising contacting the cell with aneffective amount of any of the antibodies or polypeptides of theaforementioned aspects or embodiments, as well as any other aspects orembodiments described herein.

The invention further provides a method of modulating the function ofpericytes and/or vascular smooth muscle cells, comprising administeringan effective amount of an antagonist of human Notch3 to the subject. Insome embodiments, the method inhibits angiogenesis by modulating thefunction of pericytes and/or vascular smooth muscle cells. In someembodiments, the antagonist is an antibody or polypeptide as describedin any of the aforementioned aspects or embodiments, as well as anyother aspects or embodiments described herein. In certain embodiments,the vascular development that is inhibited is aberrant vasculardevelopment. In certain embodiments, the vascular development that isinhibited is in a tumor. In certain embodiments, the method furthercomprises administering to the subject an antagonist of VEGF or of aVEGF receptor.

In addition, the invention provides methods of inhibiting angiogenesisor vascular development in a subject, comprising administering aneffective amount of a Notch antagonist to the subject. In certainembodiments, the Notch antagonist is a Notch3 antagonist. In certainembodiments, the Notch antagonist is a Notch2 antagonist. In certainembodiments, the antagonist is an antagonist of Notch2 and/or 3. In someembodiments, the antagonist is an anti-Notch2/3 antibody. In someembodiments, the methods of inhibiting angiogenesis comprisesadministering an antibody or polypeptide of any of the aforementionedaspects or embodiments, as well as any other embodiments or aspectsdescribed herein. In certain embodiments, the angiogenesis is tumorangiogenesis. In certain embodiments, the vascular development is at thesite of a tumor. In certain alternative embodiments, the angiogenesis isnot tumor angiogenesis. In certain embodiments, the inhibition ofangiogenesis or vascular development is due, at least in part, tomodulation of the function of pericytes and/or vascular smooth musclecells. In certain embodiments, the method further comprisesadministering to the subject an antagonist of vascular endothelial cellgrowth factor (VEGF) or of a VEGF receptor.

Methods of reducing the tumorigenicity of a tumor (e.g., a tumor thatcomprises cancer stem cells) are also provided. In certain embodiments,the methods comprise administering to a subject in need thereof (e.g.,subject has a tumor) a therapeutically effective amount of the Notchantagonist. In certain embodiments, the Notch antagonist is an antibodythat binds Notch2. In certain embodiments, the Notch antagonist is anantibody that binds Notch3. In certain embodiments, the Notch antagonistis an antibody that binds Notch2 and Notch3. In certain embodiments, theNotch antagonist is an antibody or polypeptide of any of theaforementioned aspects or embodiments, as well as any other embodimentsor aspects described elsewhere herein. In certain embodiments, thefrequency of cancer stem cells in the tumor is reduced by administrationof the antibody. In some embodiments, the tumor is a colorectal tumor,breast tumor, pancreatic tumor or melanoma.

It is further envisioned that the agents and antagonists of the presentinvention can be used to treat various conditions characterized byexpression of and/or increased responsiveness of cells to a Notchreceptor. The invention provides methods of treating proliferativedisease, such as cancer, diseases associated with angiogenesis (e.g.,angiogenesis-dependent diseases), and diseases in which the upregulationor deregulation of Notch signaling plays a role.

In certain embodiments the disease to be treated with the Notch-bindingagents or antagonists is a Notch-related disease. In certainembodiments, the disease is characterized by upregulation orderegulation of Notch signaling (e.g., Notch2 and/or Notch3 signaling).In certain embodiments, the disease or tumor is Notch2 and/orNotch3-dependent.

Particularly, it is envisioned that the antagonists (e.g., antibodies)against a Notch receptor will be used to treat proliferative disordersincluding, but not limited to, benign and malignant tumors of thekidney, liver, bladder, breast, stomach, ovary, colon, rectum, prostate,lung, vulva, thyroid, head and neck, brain (glioblastoma, astrocytoma,medulloblastoma, etc.), blood and lymph (leukemias and lymphomas). Incertain embodiments, the proliferative disorder that Notch-binding agentor antagonist is used to treat is colorectal cancer, breast cancer,pancreatic cancer, or melanoma. In certain embodiments, the cancercomprises cancer stem cells.

In certain embodiments, the tumors treated are solid tumors. Examples ofsolid tumors that can be treated using a therapeutic composition of theinstant invention, for example, an antibody that binds Notch include,but are not limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. The invention is applicable to sarcomas and epithelialcancers, such as ovarian cancers and breast cancers. In certainembodiments, the tumor is a colorectal tumor, breast tumor, pancreatictumor, or melanoma. In certain embodiments, the tumor is an ovariantumor. In certain embodiments, the tumor is a medulloblastoma. Incertain embodiments, the tumor comprises cancer stem cells.

In certain embodiments, the disease to be treated with the Notch-bindingagent or antagonist is a disease associated with angiogenesis. Incertain embodiments, the disease is cancer. In certain otherembodiments, the disease is not a cancerous condition. For example, thedisease may be wet macular degeneration, age related maculardegeneration, diabetic retinopathy, a hemangioma, rheumatoid arthritis,psoriasis, neovascular glaucoma, polycystic ovary disease, endometriosisand inflammatory bowel disorders.

In certain embodiments, the tumor expresses the Notch receptor orreceptors to which the Notch-binding agent or antagonist is targeted. Incertain embodiments, the tumor expresses Notch2 and/or Notch3. Incertain embodiments, the tumor overexpresses Notch2 and/or Notch3. Incertain embodiments, the tumor is dependent upon one or more Notchreceptors to which the antibody administered specifically binds. Forexample, in certain embodiments, an antibody that specifically bindsNotch2 (or Notch2 and Notch3) may be used to inhibit the growth orotherwise target the Notch2-dependent tumor. In certain embodiments, anantibody that specifically binds Notch3 (or Notch2 and Notch3) may beused to inhibit the growth or otherwise target the Notch3-dependenttumor. In certain embodiments, the tumor comprises cancer stern cells.

In certain embodiments, the tumor is homozygotic or heterozygotic for aninactivating deletion or mutation in the gene encoding the tumorsuppressor phosphatase and tensin homolog (PTEN). In certainembodiments, the tumor comprising the deletion or mutation is a breasttumor.

The antagonists are administered as an appropriate pharmaceuticalcomposition to a human patient according with known methods. Suitablemethods of administration include intravenous administration as a bolusor by continuous infusion over a period of time, by intramuscular,intraperitoneal, intravenous, intratumoral, intraarterial,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes.

In certain embodiments, in addition to administering a Notch antagonist,the method or treatment further comprises administering a secondtherapeutic agent (prior to, concurrently with, and/or subsequently toadministration of the Notch antagonist). In certain embodiments, thesecond therapeutic agent is an anti-cancer and/or anti-angiogenic agent.Pharmaceutical compositions comprising the Notch antagonist and thesecond therapeutic agent are also provided.

It will be appreciated that the combination of a Notch antagonist (e.g.,antibody) and a second therapeutic agent may be administered in anyorder or concurrently. In selected embodiments, the Notch antagonistswill be administered to patients that have previously undergonetreatment with the second anti-cancer agent. In certain otherembodiments, the Notch antagonist and the second therapeutic agent willbe administered substantially simultaneously or concurrently. Forexample, a subject may be given the Notch antagonist while undergoing acourse of treatment with the second therapeutic agent (e.g.,chemotherapy). In certain embodiments, the Notch antagonist will beadministered within 1 year of the treatment with the second therapeuticagent. In certain alternative embodiments, the Notch antagonist will beadministered within 10, 8, 6, 4, or 2 months of any treatment with thesecond therapeutic agent. In certain other embodiments, the Notchantagonist will be administered within 4, 3, 2, or 1 week of anytreatment with the second therapeutic agent. In some embodiments, theNotch antagonist will be administered within 5, 4, 3, 2, or 1 days ofany treatment with the second therapeutic agent. It will further beappreciated that the two agents or treatment may be administered to thesubject within a matter of hours or minutes (i.e., substantiallysimultaneously).

Useful classes of anti-cancer agents include, for example, antitubulinagents, auristatins, DNA minor groove binders, DNA replicationinhibitors, alkylating agents (e.g., platinum complexes such ascisplatin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, performing compounds, purine antimetabolites, puromycins,radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,vinca alkaloids, or the like. In certain embodiments, the secondanti-cancer agent is an antimetabolite, a topoisomerase inhibitor, or anangiogenesis inhibitor.

Anticancer agents that may be administered in combination with the Notchantagonists include chemotherapeutic agents. Thus, in some embodiments,the treatment involves the combined administration of an antagonist ofthe present invention and a chemotherapeutic agent or cocktail ofmultiple different chemotherapeutic agents. Treatment with an antagonistcan occur prior to, concurrently with, or subsequent to administrationof chemotherapies. Chemotherapies contemplated by the invention includechemical substances or drugs which are known in the art and arecommercially available, such as doxorubicin, 5-fluorouracil, cytosinearabinoside (Ara-C), cyclophosphamide, thiotepa, busulfan, cytoxin,taxol, methotrexate, cisplatin, melphalan, vinblastine and carboplatin.Combined administration can include co-administration, either in asingle pharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously. Preparation and dosing schedules for suchchemotherapeutic agents can be used according to manufacturers'instructions or as determined empirically. Preparation and dosingschedules for such chemotherapy are also described in ChemotherapyService Ed., M. C. Perry, Williams & Wilkins, Baltimore. Md. (1992).

Chemotherapeutic agents useful in the instant invention also include,but are not limited to, alkylating agents such as thiotepa andcyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamrine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustardssuch as chlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine: mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids such aspaclitaxel (TAXOL) and doxetaxel (TAXOTERE, Rhone); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Chemotherapeutic agents also includeanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and antiandrogens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

In certain embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapy agents thatinterfere with the action of a topoisomerase enzyme (e.g., topoisomeraseI or II). Topoisomerase inhibitors include, but are not limited to,doxorubicin HCL, daunorubicin citrate, mitoxantrone IHCL, actinomycin D,etoposide, topotecan HCL, teniposide (VM-26), and irinotecan. In certainembodiments, the second anticancer agent is irinotecan. In certainembodiments, the tumor to be treated is a colorectal tumor and thesecond anticancer agent is a topoisomerase inhibitor, such asirinotecan.

In certain embodiments, the chemotherapeutic agent is ananti-metabolite. An anti-metabolite is a chemical with a structure thatis similar to a metabolite required for normal biochemical reactions,yet different enough to interfere with one or more normal functions ofcells, such as cell division. Anti-metabolites include, but are notlimited to, gemcitabine, fluorouracil, capecitabine, methotrexatesodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside,thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine,6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, aswell as pharmaceutically acceptable salts, acids, or derivatives of anyof these. In certain embodiments, the second anticancer agent isgemcitabine. In certain embodiments, the tumor to be treated is apancreatic tumor and the second anticancer agent is an anti-metabolite(e.g., gemcitabine).

In other embodiments, the treatment involves the combined administrationof an antagonist of the present invention and radiation therapy.Treatment with an antagonist can occur prior to, concurrently with, orsubsequent to administration of radiation therapy. Any dosing schedulesfor such radiation therapy can be used.

In other embodiments, the treatment can involve the combinedadministration of antibodies of the present invention with otherantibodies against additional tumor associated antigens including, butnot limited to, antibodies that bind to the EGF receptor (EGFR) (e.g.,Erbitux®), the erbB2 receptor (HER2) (e.g., Herceptin®), and vascularendothelial growth factor (VEGF) (e.g., Avastin®). In certainalternative embodiments, the second anti-cancer agent comprises anantibody that specifically binds to human DLL4 or other ligand of aNotch receptor or an antibody that specifically binds to an additionalhuman Notch receptor. Exemplary, anti-DLL4 antibodies, are described,for example, in U.S. Patent Application Publication No. US 2008/0187532,incorporated by reference herein in its entirety. Additional anti-DLL4antibodies are described in, e.g., International Patent Publication Nos.WO 2008/091222 and WO 2008/0793326, and U.S. Patent ApplicationPublication Nos. US 2008/0014196, US 2008/0175847; US 2008/0181899; andUS 2008/0107648, each of which is incorporated by reference herein inits entirety. Exemplary anti-Notch antibodies, are described, forexample, in U.S. Patent Application Publication No. US 2008/0131434,incorporated by reference herein in its entirety. In certainembodiments, the second anti-cancer agent is an inhibitor of Notchsignaling. In certain embodiments, the second anti-cancer agent is anantibody that is an angiogenesis inhibitor (e.g., an anti-VEGFantibody). In certain embodiments, the second therapeutic agent is anantibody that specifically binds a VEGF receptor. In certainembodiments, the second therapeutic agent is AVASTIN (bevacizumab),HERCEPTIN (trastuzumab), VECTIBIX (panitumumab), or ERBITIJX(cetuximab). Combined administration can include co-administration,either in a single pharmaceutical formulation or using separateformulations, or consecutive administration in either order butgenerally within a time period such that all active agents can exerttheir biological activities simultaneously.

Furthermore, treatment can include administration of one or morecytokines (e.g., lymphokines, interleukins, tumor necrosis factors,and/or growth factors) or can be accompanied by surgical removal ofcancer cells or any other therapy deemed necessary by a treatingphysician.

For the treatment of the disease, the appropriate dosage of anantagonist of the present invention depends on the type of disease to betreated, the severity and course of the disease, the responsiveness ofthe disease, whether the antagonist is administered for therapeutic orpreventative purposes, previous therapy, patient's clinical history, andso on, all at the discretion of the treating physician. The antagonistcan be administered one time or over a series of treatments lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved (e.g., reduction in tumorsize). Optimal dosing schedules can be calculated from measurements ofdrug accumulation in the body of the patient and will vary depending onthe relative potency of an individual antagonist. The administeringphysician can easily determine optimum dosages, dosing methodologies andrepetition rates. In general, dosage is from 0.01 μg to 100 mg per kg ofbody weight, and can be given once or more daily, weekly, monthly oryearly. The treating physician can estimate repetition rates for dosingbased on measured residence times and concentrations of the drug inbodily fluids or tissues.

In certain embodiments, the patients under consideration for treatmentwith the Notch antagonist are screened prior to treatment with the Notchantagonist. In certain embodiments, a tumor in a patient or a tumor thathas been removed from a patient is tested for the presence of cancerstem cells. In certain embodiments, the tumor is tested for expressionof the one or more Notch receptors (e.g., Notch2 and/or Notch3) to whichthe antagonist binds. In certain embodiments, the tumor is tested forthe presence of an inactivating deletion or mutation in the geneencoding the tumor suppressor phosphatase and tensin homolog (PTEN). Incertain embodiments, the tumor so tested is a breast tumor.

For example, the invention provides a method of selecting a subject fortreatment with a Notch2 and/or Notch 3 antagonist, wherein the subjecthas a tumor or has had a tumor removed. In certain embodiments, themethod comprises (a) determining if the tumor comprises a deletion ormutation in the PTEN gene, and (b) selecting the subject for treatmentwith the Notch 3 antagonist if the tumor comprises the deletion ormutation.

In certain alternative embodiments of the present invention, patientsscreened for the presence of colon adenomas or polyps are tested forallelic loss and somatic mutations via a genetic test. In someembodiments the genetic test screens for loss or mutations in the Wntpathway including, for example, in APC, Axin2 or beta-catenin.

In another aspect, the present invention provides kits that can be usedto perform the methods described herein. In some embodiments, a kitcomprises an antibody or antibodies specific for a Notch receptor, apurified antibody or antibodies, in one or more containers. In someembodiments, a kit further comprises a substantially isolated Notchreceptor comprising an epitope that is specifically immunoreactive withthe antibody or antibodies included in the kit, a control antibody thatdoes not react with the Notch receptor, and/or a means for detecting thebinding of an antibody to a Notch receptor (such as, for example, afluorescent chromophore, an enzymatic substrate, a radioactive compoundor a luminescent compound conjugated to the antibody against a Notchreceptor or to a second antibody that recognizes the antibody against aNotch receptor). In other embodiments, a kit comprises reagents specificfor the detection of mRNA or cDNA (e.g., oligonucleotide probes orprimers) of one or more Notch receptor. In some embodiments, the kitscontain all of the components necessary and/or sufficient to perform adetection assay, including all controls, directions for performingassays, and any necessary software for analysis and presentation ofresults.

A compartment kit includes any kit in which reagents are contained inseparate containers. Such containers include small glass containers,plastic containers or strips of plastic or paper. Such containers allowsone to efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the antibodies or probes used in the methods,containers which contain wash reagents (such as phosphate bufferedsaline, Tris-buffers, etc.), and containers which contain the reagentsused to detect the bound antibody or probe. One will readily recognizethat the disclosed polynucleotides, polypeptides and antibodies of thepresent invention can be readily incorporated into one of theestablished kit formats which are well known in the art.

In some embodiments, the invention further provides a kit comprising aNotch-binding agent or antagonist and a second therapeutic agent. Incertain embodiments, the Notch-binding agent or antagonist is anantibody that specifically binds to Notch 2 and/or Notch3. In certainembodiments, the Notch-binding agent or antagonist is an antibody thatspecifically binds to Notch 2 and Notch3. In certain embodiments, thesecond therapeutic agent is an anti-cancer agent and/or ananti-angiogenic agent.

In one aspect, the present invention provides a method of identifying amolecule that binds to a non-ligand binding region of an extracellulardomain of a human Notch receptor and inhibits tumor growth, the methodcomprising: i) incubating the molecule with the non-ligand bindingdomain of the extracellular domain of the human Notch receptor; ii)determining if the molecule binds to the non-ligand binding region ofthe extracellular domain of the human Notch receptor; and iii)determining if the molecule inhibits tumor growth. Molecules thatspecifically bind a non-ligand binding region of an extracellular domainof a human Notch receptor include, but are not limited to, small organicmolecules, polypeptides, and antibodies.

Screening can be performed using any suitable method known in the art.In certain embodiments, screening is performed in vitro. In someembodiments, cells expressing a non-ligand binding region of theextracellular domain of a human Notch receptor are incubated with alabeled molecule and specific binding of the labeled molecule to anon-ligand binding region of the extracellular domain of a human Notchreceptor is determined by FACS analysis. In some embodiments, anon-ligand binding region of the extracellular domain of a human Notchreceptor is expressed by phage display, and molecules that specificallybind to a non-binding region of the extracellular domain of a humanNotch receptor are identified. Other suitable methods for identifyingmolecules that specifically bind to a non-ligand binding region of ahuman Notch receptor include, but are not limited to, ELISA, Western (orimmuno) blotting, and yeast-two-hybrid.

Molecules that specifically bind to a non-ligand binding region of anextracellular domain of a human Notch receptor are then tested forinhibition of tumor cell growth. Testing can be performed using anysuitable method known in the art. In certain embodiments, molecules thatspecifically bind to non-ligand binding region of the extracellulardomain of a human Notch receptor are tested for the ability to inhibittumor growth in vitro. In some embodiments, molecules that specificallybind a non-ligand binding region of the extracellular domain of a humanNotch receptor are incubated with tumor cells in culture andproliferation of tumor cells in the presence of a molecule thatspecifically binds a non-ligand binding region of the extracellulardomain of a human Notch receptor is determined and compared to tumorcells incubated with a non-binding control molecule. In certainembodiments, molecules that specifically bind to non-ligand bindingregion of the extracellular domain of a human Notch receptor are testedfor the ability to inhibit tumor growth in vivo. In certain embodiments,molecules that specifically bind a non-ligand binding region of theextracellular domain of a human Notch receptor are injected into ananimal xenograft model and the growth of tumors in animals treated withmolecules that specifically bind to non-ligand binding region of theextracellular domain of a human Notch receptor is determined andcompared to animals treated with a non-binding control molecule.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

Example 1 Production of Human Antibodies to Notch2

Human antibodies that specifically recognize the non-ligand bindingportion of the extracellular domain of a Notch2 receptor were isolatedusing phage display technology. A synthetic antibody library containinghuman antibody variable domains was screened for specific and highaffinity recognition of a Notch2 receptor.

Briefly, 2×10¹³ Fab displaying phage particles were incubated with apassively immobilized, recombinant Notch2 Fc fusion protein (SEQ ID NO:21) comprising the extracellular ligand binding site of Notch2 andsurrounding EGF repeats (EGF1-12) in round one. The non-specific phagewere washed off, and then the specific phage were eluted with DTT. Theeluted output was used to infect TG1 F+ bacteria, rescued with helperphage, and then Fab display induced with IPTG (0.25 mM). This processwas repeated for two additional rounds and then round three was screenedin ELISA against passively immobilized recombinant Notch2 (EGF1-12) Fcfusion (5 μg/ml).

A particular Fab (59R1) was identified that bound the human Notch2receptor and blocked binding of Jagged1 to human Notch2. Binding of the59R1 Fab to human Notch2 was verified by FACS assay using a stable humancell line HEK-293 which overexpressed human Notch2 (hN2) (FIG. 1A). Fabbinding was detected by phycoerythrin (PE)-conjugated goat anti-humanFab (Jackson Immunochemicals). The 59R1 Fab (referred to in FIG. 1A asclone 1) demonstrated good binding to hN2. The 59R1 Fab alsodemonstrated good blocking activity against the Notch ligand humanJagged1 in a binding assay using the same stable cell line (FIG. 1B).Ligand binding and blocking was determined by incubating hJagged1extracellular domain (ECD) fused to human Fc constant region with thecells and Fabs selected from the phage library and using PE-conjugatedgoat anti-human Fc gamma specific antibodies (Jackson immunochemicals)for detection.

The sequences of the VH and VL of the 59R1 Fab are provided in SEQ IDNO: 11 and SEQ ID NO: 12 (including N-terminus bacterial signalsequences that are cleaved upon secretion), respectively. The CDRs ofthe 59R1 Fab are as indicated in Table 2 below.

TABLE 2  CDRs of 59R1 human Fab and IgG antibodies Heavy ChainLight Chain Lea 

CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 59R 

SSSGMS VIASSGSNTYYADSVK 

GIFFA 

RASQSVRSNYL 

GASSRA QQYSNFPI (SEQ ID (SEQ ID NO: 6) (SEQ ID (SEQ ID NO: 8) (SEQ ID(SEQ ID NO: 5) NO: 7) NO: 9) NO: 10)

indicates data missing or illegible when filed

Variable regions based on those of the 59R1 Fab were cloned into Igexpression vectors containing human IgG2 heavy-chain and kappalight-chain along with their respective mammalian signal sequences forexpression in Chinese Hamster Ovary (CHO) cells. The VH and VL of the59R1 IgG antibody are provided as SEQ ID NO: 13 and SEQ ID NO. 14,respectively. The amino acid sequence of the heavy chain and light chainof the 59R1 IgG antibody (including signal sequences) are provided asSEQ ID NO: 16 and SEQ ID NO: 18, respectively. The signal sequence atthe N-terminus of the amino acid sequence of each of the chains iscleaved upon secretion. The nucleic acid sequences encoding the heavyand light chains of the 59R1 IgG antibody are provided as SEQ ID NO: 1and SEQ ID NO: 3, respectively. Protein A purification was used topurify the antibodies. Bacterial plasmid DNA containing a synthetic DNAinsert encoding the heavy and light chain of the 59R1 IgG2 antibody DNAwas deposited as “59R1” with the ATCC, 10801 University Boulevard,Manassas, Va., USA, under the conditions of the Budapest Treaty on Oct.15, 2008, and assigned designation number PTA-9547.

In addition, the 59R1 IgG2 antibody was assayed for its ability to blockbinding of DLL4 to the human Notch 2 receptor by FACS analysis. HEK-293cells stably overexpressing human Notch2 were incubated with theantibody at various concentration and then detected for hNotch2 binding(FIG. 1C) by PE-conjugated goat anti-human Fc gamma specific antibody,or ligand blocking activity (FIG. 1D). Ligand blocking was determined byincubating the cells with human DLL4 ECD tagged with the rabbit Fcconstant region and the 59R1 antibody at a range of concentrations, andthen detecting the hDLL4 by PE-conjugated donkey anti-rabbit antibody.Binding of hNotch2 and ligand blocking activity were thus confirmed forthe 59R1 IgG2 antibody.

A germlined variant of 59R1 (referred to herein as “59RGV”) was alsoexpressed and purified. The VH and VL of the 59RGV antibody are providedas SEQ ID NO: 19 and SEQ ID NO: 20, respectively. The amino acidsequence of the heavy chain and light chain of the 59RGV antibody(including signal sequences) are provided as SEQ ID NO: 2 and SEQ ID NO:4, respectively. The signal sequence at the N-terminus of the amino acidsequence of each of the chains is cleaved upon secretion. The nucleicacid sequences encoding the heavy and light chains of the 59RGV antibodyare provided as SEQ ID NO: 15 and SEQ ID NO: 17, respectively.

Highly hydrophobic CDRs have the potential, in certain instances, toallow for unfavorable, non-specific binding by an antibody. Since theamino acid sequence of the heavy chain CDR3 of 59R1 had an unusualdegree of hydrophobic character, variants of 59R1 that contained heavychain CDR3 sequences with decreased hydrophobic character were produced.Heavy chain CDR3 affinity maturation was conducted by allowingrestricted changes from the parental sequence (GIFFAI; SEQ ID NO:7) asshown in FIG. 1E. Allowed amino acids at each position were allowed tochange from parental residues to the residues indicated in FIG. 1E.Improved variants were isolated by screening them for improved JAG1blocking ability as shown in FIG. 1F (indicated with arrows). Briefly,Fabs (1 and 10 μg/ml) were mixed with hJAG1-rb Fc (preclustered 5 μg/mlto 2 μl/ml PE-conjugated donkey anti-rabbit) and then added to hNotch2stably transfected 293 cells. hJAG1 binding was then assessed using flowcytometry. Six improved variants (versus 59R1 Fab) were isolated andtheir HC CDR3 sequences were as follows: SIFYPT (SEQ ID NO:22), SSFFAS(SEQ ID NO:23), SSFYAS (SEQ ID NO:24), SSFFAT (SEQ ID NO:25), SIFYPS(SEQ ID NO:26), and SSFFAN (SEQ ID NO:27). The sequences of the heavychain variable regions for these variants are sequences SEQ ID NO:52,SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQ IDNO:57.

Example 2 Cross-Reactivity and Binding Affinity of Anti-Notch2/3 59R1Antibody

The ability of the 59R1 IgG2 antibody to cross-react with other Notchreceptors was determined by FACS assay using HEK-293 cells transientlytransfected with the human Notch1, Notch2, Notch3 or Notch4 expressionplasmid and green fluorescent protein (GFP) as a transfection control.GFP positive cells indicated expression of the transgene. The 59R1 IgG2was added to the cells at 2 μg/ml and detected by PE-conjugated goatanti-human Fc gamma specific (Jackson Immunochemicals). All Notchconstructs were full length. The results are shown in FIG. 2. As shownin FIG. 2, the 59R1 IgG2 antibody specifically binds to both humanNotch3 and human Notch2, but does not bind significantly to full-lengthhuman Notch1 or human Notch4.

The affinities for human and mouse Notch1, Notch2, Notch3, and Notch4were determined using a Biacore 2000 instrument. Briefly, recombinanthuman and mouse Notch proteins (EGF10-15 for Notch1, 2, & 4; EGF9-14 forNotch3) were immobilized on a CM5 chip using standard amine basedchemistry (NHS/EDC). For hNotch2, EGF1-12 was also tested for binding.Different antibody concentrations (1-100 nM) were injected over theprotein surfaces and kinetic data were collected over time. The data wasfit using the simultaneous global fit equation to yield dissociationconstants (K_(D), nM) for each Notch (Table 3).

TABLE 3 59R1 IgG Dissociation Constants (K_(D)) mNotch1 hNotch1 mNotch2hNotch2 hNotch2 mNotch3 hNotch3 hNotch4 Ab (nM) (nM) (nM) (nM)* (nM)**(nM) (nM) (nM) 59R1 >10 86.4 0.35 <0.1 <0.1 0.13 0.12 NB *N2(EGF1-12)**N2(EGF 10-155)

Example 3 Epitope Mapping of Anti-Notch2/3 Antibody 59R1

To identify antibodies that recognize specific non-ligand bindingregions of the Notch receptor extracellular domains, epitope mapping wasperformed.

The binding of anti-Notch2/3 antibodies to supernatant from 1HEK 293cells transfected with sequences encoding recombinant human Notch2 Fcfusion proteins comprising the full length human Notch2 protein orvarious human Notch2 deletion constructs containing various deletions ofEGF repeats one to twelve were tested by ELISA. See Table 4 below.HEK-293 cells were transiently transfected with pcDNA 3.1 (invitrogen)with hNotch2 cDNA's encoding for the indicated amino acids fused to theconstant region of human IgG (hFc). Supernatants were harvested 48 hourslater. To capture hN2-hfc proteins, 96 well plates were first coatedwith goat anti-human Fc gamma specific IgG (Jackson Immunochemicals,#109-006-098) at 100 ng per well in sodium bicarbonate buffer overnightat 4°. Plates were washed and blocked in 5% bovine serum/PBS-Tween-20.Supernatants were added to plates and incubated at room temperature for1 hour. Plates were washed in PBS-T. 59R1 Fab was added at 10 μg/ml in5% serum/PBS-T and incubated at room temperature for 1 hour. Plates werewashed in PBS-T. Fab binding was detected by goat anti-human Fabspecific antibody conjugated to horseradish peroxidase (Thermo, #31414)diluted 1:5000 in 5% serum/PBS-T for 1 hour at room temperature. Plateswere washed and developed with 1 Step Ultra TMB (Thermo, #34028). Plateswere read on a Perkin Elmer Victor 1420 plate reader. Anti-Notch2/3 59R1antibody bound only to supernatant from cells expressing recombinantNotch2 proteins comprising EGF 10, which consists of amino acids 375-417of human Notch 2. (FIG. 3A).

TABLE 4 Human Notch2 deletion constructs Construct amino acids hN2 1-3 1-144 hN2 1-4  1-181 hN2 1-5  1-221 hN2 1-6  1-263 hN2 1-7  1-301 hN21-8  1-341 hN2 1-9  1-378 hN2 1-10  1-417 hN2 1-11  1-456 hN2 1-12 1-493 hN2 8-12 296-493 hN2 9-12 376-493 hN2 10-12 375-493 hN2 11-12413-493 hN2 12-12 454-493

Moreover, FACS analysis shows that 59R1 Fab antibody binding wasretained when EGF11 or EGF12 were deleted from full length Notch2recombinant protein expressed by HEK 293 cells (FIG. 3B). Pointmutations were made within EGF10 of Notch2 fusion proteins and bindingof 59R1 to each EGF10 mutant was determined by FACS analysis. HEK-293cells were transiently transfected with the indicated Notch expressionplasmid and GFP as a transfection control. GFP positive cells indicatedexpression of the transgene. The 59R1 Fab antibody was added to thecells at 10 μg/ml and detected by PE-conjugated goat anti-human (JacksonImmunochemicals).

To verify that loss of EGF repeat 10 does not interfere with ligandbinding, a mutant hNotch2 missing amino acids 375-412 was generated andtested for binding to 59R1, a hNotch2 monoclonal 59M70 directed againstEGF 1-4, and binding to the ligand human DLL4 (FIG. 3C). FACS analysisof HEK-293 cells transiently transfected with the indicated Notchexpression plasmid and GFP as a transfection control. GFP positive cellsindicate expression of the transgene Anti-Notch2 (59M70) was added at 20μg/ml and detected by PE-conjugated goat anti-mouse (Caltag, #3004-4).59R1 (IgG2) was added to the cells at 2 μg/ml and detected byPE-conjugated goat anti-human Fc gamma specific (Jacksonimmunochemicals). Ligand binding was determined by incubation of thecells with human DLL4 extracellular domain (ECD) fused to rabbit IgGconstant region at 5 μg/ml and detected by PE-conjugated donkeyanti-rabbit. As shown in FIG. 3C, ligand and 59M70 both bind to hNotch2in the absence of EGF 10, but 59R1 does not.

A comparison analysis of the EGF 10 regions of human Notch1, Notch2, andNotch4 and the EGF 9 region of human Notch3 (the equivalent of EGF 10 inthe other Notch receptors) was performed to determine likely bindingsites for 59R1 (FIG. 14A). As a result of the analysis, several pointmutants were created within full-length Notch2, converting residueswithin EGF10 to the corresponding amino acids in human Notch 1. Also,conversely, point mutations were made in hNotch1 EGF 10 convertingresidues to the corresponding hN2 residues. Mutants in full-length Notchsequences were generated by QuikChange® mutagenesis (Stratagene) andverified by sequencing. Binding to the mutants was determined by FACSanalysis (FIGS. 14B and 14C). 59R1 was detected by PE-conjugated goatanti-human Fc gamma specific antibody (Jackson Immunochemicals,#109-116-170). The amino acids necessary for 59R1 binding to hNotch 2were thus determined to be histidine 385, alanine 388, and leucine 389(residues within the boxed hNotch2 sequence shown in FIG. 14A). Thecorresponding residues in hNotch3 are histidine 361, alanine 364, andisoleucine 365.

Example 4 Anti-Notch2/3 Antibody 59R1 Inhibits Notch2 Signaling

Luciferase reporter assays were used to assay the 59R1 antibody for itsability to block hDLL4-, hJAG1-, and hJAG2-induced Notch2 signaling.

Hela cells that stably overexpress human Notch2 were transientlytransfected with firefly luciferase with a synthetic 8×CBS promoter (Onget al., 2006, J. of Biological Chemistry, 281:5106-5119), pSPORT6MAML-1, and Renilla luciferase-CMV as a transfection control. Cells wereincubated with 100 ng of immobilized hDLL4 (R&D systems) with theindicated antibodies for 16 hours and then assayed using Dual-Glo(Promega) according to the manufacturer's instructions. Control antibodywas at a concentration of 40 μg/ml. 59R1 IgG2 antibody was titrated,starting at 40 μg/ml, and then diluted by one-fourth. The gammasecretase inhibitor (GSI) dibenzazepine (DBZ) was used as a control at 1μM. As shown in FIG. 4A, the 59R1 antibody was found to inhibithDLL4-induced Notch2 reporter activity.

Hela cells that stably overexpress human Notch2 were transientlytransfected with firefly luciferase with a synthetic 8×CBS promoter,pSPORT6 MAML-1, and Renilla luciferase-CMV as a transfection control.Cells were incubated with either 200 ng of immobilized hJAG 1 (R&Dsystems) or hJAG2 (R&D systems) for 16 hours and then assayed usingDual-Glo (Promega) according to the manufacturer's instructions. 59R1IgG2 antibody was at a concentration of 40 μg/ml. The gamma secretaseinhibitor (GSI) dibenzazepine (DBZ) was used as a control at 1 μM. Asshown in FIGS. 4B and 4C, the 59R1 antibody was found to inhibit bothhJAG1- and hJAG2-induced Notch2 reporter activity, respectively.

Example 5 Anti-Notch2/3 Antibody 59R1 Prevents In Vivo Tumor Growth

This example describes the use of an anti-Notch2/3 receptor antibody(59R1) that binds a non-ligand binding region of the Notch receptors(EGF10 of Notch2 and EGF10 of Notch3) to prevent tumor growth in axenograft model.

In certain embodiments, NOD/SCID mice injected with 50,000 PE13 or T3breast tumor cells were treated with anti-Notch2/3 antibody 59R1 orcontrol antibody 1B7.11 two days following cell injections. Antibodieswere dosed at 10 mg/kg twice week. Anti-Notch2/3 antibody 59R1significantly reduced both PEI 3 (FIG. 5A) and T3 (FIG. 5B) tumor growthcompared to control.

Example 6 In Vivo Treatment of Tumors Using Anti-Notch 2/3 Antibody 59R1

This example describes the use of anti-Notch 2/3 antibodies to treatcancer in a xenograft model.

In one experiment, the 1×10 viable Colo-205 colon tumor cells wereinjected into 6-8 week-old immunodeficient hg/u XID female mice on aSwiss CD-1 background. Tumors were allowed to grow to a size of between65 to 200 mm³ after which mice were randomized (n=10 per experimentalgroup), and antibodies administration begun. Animals were treated with15 mg/kg of either control 1B7.11 antibodies or anti-Notch2/3 59R1antibodies once weekly. Tumor size was measured twice weekly, and tumorvolume was calculated as described (see Michieli et al., 2004, CancerCell, 6:61-73). Anti-Notch2/3 antibody 59R1 significantly reducedColo-205 tumor growth compared to control (FIG. 5C).

In another experiment, anti-Notch2/3 antibodies were tested for aneffect on pancreatic tumor growth. NOD/SCID mice were injected with30,000 PN4 pancreatic tumor cells sub-cu in the right flank, and tumorswere allowed to grow until they had reached an average volume of 100mm³. Animals were randomized and dosing of anti-Notch2/3 antibody 59R1or control antibody 1B711 was initiated. Antibodies were dosed at 15mg/kg given once per week. Anti-Notch2/3 antibody 59R1 significantlyreduced PN4 tumor growth compared to control (FIG. 5D).

In a further experiment, anti-Notch2/3 antibodies were tested for aneffect on breast tumor growth. NOD/SCID mice were injected with 50,000PE13 or T3 breast tumor cells, and tumors were allowed to grow to a sizeof between 65 to 200 mm³ after which mice were randomized (n=10 perexperimental group), and antibodies administration begun. Animals weretreated with 15 mg/kg of either control 1B7.11 antibodies oranti-Notch2/3 59R1 antibodies twice weekly. Tumor size was measuredtwice weekly, and tumor volume was calculated as described (see Michieliet al., 2004). Anti-Notch2/3 antibody 59R1 significantly reduced growthof both PE13 (FIG. 5E) and TE (FIG. 5F) tumors compared to control.

At the end point of antibody treatment, tumors may be harvested forfurther analysis. In some embodiments, a portion of the tumor isanalyzed by immunofluorescence to assess antibody penetration into thetumor and tumor response. A portion of each harvested tumor fromanti-Notch2/3 antibody treated and control antibody treated mice isfresh-frozen in liquid nitrogen, embedded in O.C.T., and cut on acryostat as 10 μm sections onto glass slides. Alternatively a portion ofeach tumor is formalin-fixed, paraffin-embedded, and cut on a microtomeas 10 μm section onto glass slides. Sections are post-fixed andincubated with chromophore labeled antibodies that specificallyrecognize injected antibodies to detect anti-Notch2/3 antibody orcontrol antibodies present in the tumor biopsy. Furthermore, antibodiesthat detect different tumor and tumor recruited cell types such as, forexample, anti-VE cadherin (CD144) or anti-PECAM-1 (CD31) antibodies todetect vascular endothelial cells, anti-smooth muscle alpha-actinantibodies detect vascular smooth muscle cells, anti-Ki67 antibodies todetect proliferating cells, TUNEL assays to detect dying cells, andanti-intracellular domain (ICD) Notch fragment antibodies to detectNotch signaling can be used to assess affects of antibody treatment onangiogenesis, tumor growth, and tumor morphology.

The effect of anti-Notch2/3 antibody treatment on tumor cell geneexpression may also be assessed. Total RNA is extracted from a portionof each harvested tumor from Notch2/3 antibody treated and controlantibody treated mice and used for quantitative RT-PCR. Expressionlevels of Notch2/3, components of the Notch2 and/or Notch3 signalingpathway, as well as cancer stern cell markers including, for example,CD44, are analyzed relative to the house-keeping gene GAPDH as aninternal control. Changes in tumor cell gene expression upon Notch2/3antibody treatment are thus determined.

In addition, the effect of anti-Notch2/3 antibody treatment on thepresence of cancer stem cells in a tumor may be assessed. Tumor samplesfrom Notch 2/3 antibody versus control antibody treated mice are cut upinto small pieces, minced completely using sterile blades, and singlecell suspensions obtained by enzymatic digestion and mechanicaldisruption. Dissociated tumor cells are then analyzed by FACS analysisfor the presence of tumorigenic cancer stem cells based on ESA+, CD44+,CD24−/low, Lin− surface cell marker expression as described in detailabove.

The tumorigenicity of cells isolated based on ESA+, CD44+, CD24−/low,Lin− expression following anti-Notch2/3 antibody treatment can thenassessed. In one example, 5,000, 1,000, 500, and 100 isolated ESA+,CD44+, CD24−/low, Lin− cancer stem cells from Notch 2/3 antibody treatedversus control antibody treated mice are re-injected subcutaneously intothe mammary fat pads of NOD/SCID mice. The tumorigenicity of cancer stemcells based on the number of injected cells required for consistenttumor formation is thus determined.

Example 7 Anti-Notch 2/3 Antibody 59R1 Delays Tumor Recurrence In VivoFollowing Paclitaxel Treatment

B51 breast tumor cells (50,000 cells per mouse) were injectedsub-cutaneously into the mammary fat pad of NOD-SCID mice. Tumors wereallowed to grow for 50 days until they had reached an average volume of˜100 mm³. Animals were randomized (n=10/group) and treatments wereinitiated. One group received a control antibody (1B711) at 10 mg/kgtwice per week and paclitaxel (Taxol) at 15 mg/kg twice per week and theother group received 59R1 at 10 mg/kg twice per week and paclitaxel at15 mg/kg twice per week. Tumor volumes were measured on the indicateddays. Treatments were carried out for 38 days until the tumor volumeshad regressed to ˜50 mm³, after which the paclitaxel treatments werehalted and the antibody treatments continued for the duration of theexperiment.

The results are shown in FIG. 6. Tumors were observed to recur morerapidly in the control group compared with the group treated with 59R1.

Example 8 Anti-Notch 2/3 Antibody 59R Decreases the Frequency of CancerStem Cells in a Tumor in Vivo

Limiting dilution assays (LDAs) can be used to assess the effect of aNotch-binding agent on solid tumor cancer stem cells and on thetumorigenicity of a tumor comprising the cancer stem cells. The assayscan be sued to determine the frequency of cancer stem cells in tumorsfrom animals treated with the Notch-binding agent or other agent and tocompare that frequency to the frequency of cancer stem cells in tumorsfrom control animals.

An LDA was used to assess the effect on the tumorigenicity of the B51breast tumors that were treated with the combination of control antibody(1B711) plus paclitaxel (Taxol) or treated with the combination of 59R1and paclitaxel, as described above in Example 7. In addition, the effectof treatment of B51 breast tumors with the control antibody alone or59R1 alone was also determined by LDA. The doses of antibodies andpaclitaxel and the schedule of dosing for the control antibody group andthe 59R1 group were the same as described in Example 7, above for theother two treatment groups. After three doses of antibodies and/orpaclitaxel, tumors were harvested, processed and dissociated into singlecells. The human tumor cells were isolated from the xenograft tumorcells by incubation with biotinylated mouse antibodies (α-mouseCD45-biotin 1:200 dilution and rat α-mouse H2 Kd-biotin 1:100 dilution,BioLegend, San Diego, Calif.) on ice for 30 min, followed by addition ofstreptavidin-labeled magnetic beads and removal of the mouse cells withthe aid of a magnet. The human cells in the suspension were harvestedand counted.

A serial titration of cells (30, 90, 270, and 810 cells) from each ofthe four treatment groups was injected in a 1:1 (v/xv) mixture of FACSbuffer and Matrigel into a new set of NOD-SCID mice (n=10/group). Tumorswere allowed to grow for 72 days. The percentage of mice with detectabletumors was determined in all groups. The cancer stem cell frequency wasthen calculated using L-Calc™ software (StemCell Technologies Inc.;downloadable from www.stemcell.com search/default.asp).

The results are shown in FIG. 7. The frequency of cancer stein cells inthe tumor in the control-treated mice (“Control”) was determined to be1:66. The frequency of cancer stem cells in the tumor in thepaclitaxel-treated mice (“Taxol”) was shown to be 1:25, indicating thattreatment with paclitaxel had actually increased the frequency of cancerstem cells in the tumor by more than two-fold relative to the control.Treatment with the 59R1 antibody, either alone (“59R1”) or incombination with paclitaxel (“Taxol+59R1”), on the other hand, reducedthe frequency of cancer stem cells in the tumors. The 59R1 antibodyalone reduced the cancer stem cell frequency in the breast tumors bymore than two-fold relative to the control. Treatment with thecombination of 59R1 antibody and paclitaxel reduced the frequency ofcancer stem cells in the tumor by more than about two-fold relative totreatment with 59R1 alone (p<0.0001), by about 4.5-fold relative totreatment with the control antibody, and by about twelve-fold relativeto treatment with paclitaxel alone. These results indicate thattreatment with the 59R1 antibody is effective at reducing thetumorigenicity of a breast tumor, whether given alone or in combinationwith paclitaxel, even though treatment with paclitaxel alone has theopposite effect.

Example 9 Additional In Vivo Treatment of Tumors Using Anti-Notch 2/3Antibody 59R1

PN4 pancreatic tumor cells (50,000 cells per mouse) were injectedsubcutaneously into the flank region of Nod-Scid mice. Tumors wereallowed to grow for 27 days until they had reached an average volume of˜120 mm³. Animals were randomized into four treatment groups(n=10/group) and treatments were initiated. One group received a controlantibody (1B711) at 10 mg/kg twice per week; one group receivedgemcitabine at 40 mg/kg once per week plus the control antibody at 10mg/kg twice per week; one group received 59R1 at 10 mg/kg twice perweek, and the fourth group received the combination of 59R1 at 10 mg/kgtwice per week and gemcitabine 40 mg/kg once per week. Tumor volumeswere measured on the indicated days. The results are shown in FIG. 8.Tumor growth was found to be inhibited by the combination of 59R1 andgemcitabine (p<0.001).

M4 melanoma tumor cells (10,000 cells per mouse) were injectedsubcutaneously into the flank region of NOD-SCID mice. Tumors wereallowed to grow for 25 days until they had reached an average volume of˜80 mm³. Animals were randomized into treatment groups (n=10/group) andtreatments were initiated. One group received a control antibody (1B711)at 10 mg/kg twice per week and one group received 59R1 at 10 mg/kg twiceper week. Tumor volumes were measured on the indicated days. The resultsare shown in FIG. 9. Tumor growth was found to be inhibited by 59R1.

C28 colon tumor cells (10,000 cells per mouse) were injectedsubcutaneously into the flank region of NOD-SCID mice. Tumors wereallowed to grow for 24 days until they had reached an average volume of˜130 mm³. Animals were randomized into four treatment groups(n=10/group) and treatments were initiated. One group received a controlantibody (1B711) at 10 mg/kg twice per week; one group receivedirintoecan at 7.5 mg/kg once per week plus the control antibody at 10mg/kg twice per week; one group received 59R1 at 10 mg/kg twice perweek, and the fourth group received the combination of 59R1 at 10 mg/kgtwice per week and irinotecan at 7.5 mg/kg once per week. Tumor volumeswere measured on the indicated days. The results are shown in FIG. 10.Tumor growth was found to be inhibited by 59R1 alone relative to thecontrol antibody group and by the combination of 59R1 and irinotecanrelative to the irinotecan group.

The 59R1 IgG2 antibody was also tested in vivo in the breast tumorxenograft lines OMP-B34, OMP-B39, OMP-B44, PE13, and UM-T1, the pancreastumor xenograft line OMP-PN8, and the colon tumor xenograft line OMP-C8.These tumor xenograft lines were established by adhering to proceduresdescribed in Al-Hajj et al., 2003, Proc. Natl. Acad. Sci. USA,100:3983-3988. Female NOD/SCID immuno-compromised mice 7-10 weeks oldwere used for the establishment of the breast tumor xenografts and maleNOD/SCID mice were used for the OMP-Pn8 and OMP-C8 tumor-models (Harlan,Indianapolis, Ind.). The 59R1 IgG2 antibody was also tested in vivo in aColo-205 colon tumor xenograft model. Female immunodeficient bg/nu XIImice on a Swiss CD-1 background were used for the Colo-205 xenografttumor model. In case of the breast cancer models, slow-releasingestrogen pellets (0.36 mg) had to be implanted. Mice were subcutaneouslyinjected on the right flank with 50,000 (OMP-B34, OMP-B39, OMP-B44,PE13, and UM-T1) or 1×10⁷ (Colo-205) viable cells, respectively, in amixture of PBS (without magnesium or calcium) and Matrigel at a 1:1ratio. The injected total volume per mouse was 200 μl with 50% beingMatrigel. Once the tumor had reached a size between 65-200 mm³, the micewere randomized. Antibodies were administered weekly and tumors measuredtwice weekly. LZ1 (a human antibody that recognizes lyzozyme) or 1B711(a murine IgG1 antibody that recognizes the hapten trinitrophenol) wasused as a control antibody for treatment of each tumor type. Tumorvolume was calculated as described Al-Hajj et al. (2003). Data areexpressed as the mean and the mean±S.E.M. Group means were comparedusing Student's two-tailed, unpaired t-test. Probability (P) values of<0.05 were interpreted as significantly different. All statisticalanalysis was performed using Microsoft EXCEL and Graph Pad PRISM.

The results of the additional in vivo experiments in Colo205, C8, PNA,B34, B39, B44, PE13, and T1 xenograft models are shown in FIGS. 11A-11H,respectively. As shown in FIG. 11A, monotherapy with the 59R1 antibodysignificantly inhibited growth of the Colo205 tumor relative to thecontrol antibody (LZ1) (p<0.01). Combination therapy with 59R1 plus theanti-VEGF antibody bevacizumab (AVASTIN) provided an even greaterinhibition of tumor growth (p<0.001) than either 59R1 or bevacizumabalone. In another colorectal xenograft model, C8, 59R1 was likewiseshown to inhibit tumor growth relative to LZ1 control antibody (FIG.11B). Similarly, 59R1 was found to inhibit pancreatic tumor growth(relative to control antibody) in the PN8 xenograft model (FIG. 11C).59R1 was also shown to inhibit breast cancer growth relative to acontrol antibody in each of the five breast cancer xenograft models B34(FIG. 11D), B39 (FIG. 11E), B44 (FIG. 11F), and PE13 (FIG. 11G). The59R1 antibody was likewise found to be effective at inhibiting tumorgrowth in the T1 breast cancer model (FIG. 11H), although it was onlyeffective in the presence of estrogen, despite T1 being an estrogenreceptor negative tumor.

Example 10 Effect of Treatment with Anti-Notch2/3 Antibody 59R1 on GeneRegulation in Xenograft Tumor Models

Gene expression levels in various xenograft tumor models treated withthe 59R1 IgG2 antibody were analyzed by microarray analysis. Global geneexpression profiling analysis was performed on Affymetrix HG-UI33 plus2.0 microarray (Affymetrix, Santa. Clara, Calif.). Three independent RNAsamples of xenograft whole tumors from the control and treatment groupswere isolated and hybridized to the microarrays according to themanufacturer's instructions. Scanned array background adjustment andsignal intensity normalization were performed with GCRMA algorithm inthe open-source bioconductor software (www.bioconductor.org). Theexpression level of each gene was normalized by z-score transformationacross the samples in the control (CTRL) and treatment (59R1) groups.Genes differentially expressed (p<0.05 and fold change >2.0) between thetwo groups were identified with Bayesian t-test (Baldi et al., 2001,Bioinformatics, 17:509-519. The expression patterns of selectedassociated differentially regulated genes in selected tumor xenograftmodels (Colo205, B44, PE13, and T1) are shown in Table 5 below. TheP-value (PVal) of each gene is the probability of significant regulationof the gene by 59R1 by chance using Bayesian t-test. A number of genesincluding the genes encoding regulator of G-protein signaling 5 (RGS5),Notch3, and hairy/enhancer-of-split related with YRPW motif-like (HEYL)protein were shown to be significantly down-regulated in the stroma ofthe 59R1-treated mice relative to the control mice. (By contrast, theseparticular genes encoding RGSS, Notch3, and HEYL. were not found to besignificantly down-regulated in the human cells of the tumors.)

TABLE 5 Differentially expressed genes in stroma of 59R1-treated tumorsColo205 B44 PE13 T1 Gene Fold pVal Fold pVal Fold pVal Fold pVal1420942_s_at −5.52 7.65E−07 −2.43 5.59E−04 −4.23 2.86E−05 −1.18 9.82E−04(Rgs5) 1417466_at −3.39 6.62E−07 −2.22 3.11E−04 −4.03 1.31E−10 −1.994.11E−04 (Rgs5) 1420941_at −5.10 1.66E−03 −2.09 1.18E−03 −2.99 1.35E−05−1.97 2.07E−03 (Rgs5) 1421964_at −3.26 3.70E−06 −2.03 2.30E−03 −1.911.67E−03 −1.01 8.86E−01 (Notch3) 1416286_at −3.08 2.69E−03 −1.573.84E−02 −1.83 6.71E−05 −1.13 4.47E−01 (Rgs4) 1434141_at −2.49 2.87E−03−1.74 1.07E−02 −4.18 1.49E−07 1.20 5.95E−01 (Gucy1a3) 1459713_s_at −1.901.90E−03 −1.70 1.01E−02 −7.28 9.89E−10 −2.14 1.79E−04 (Tmem16a)1420872_at −1.94 1.90E−02 −1.65 7.68E−03 −3.06 8.52E−10 −1.01 7.13E−01(Gucy1b3) 1422789_at −1.73 1.20E−02 −4.92 2.42E−08 −2.17 1.58E−04 −2.169.27E−04 (Aldh1a2) 1419302_at −3.28 5.61E−03 −1.12 2.36E−01 −1.775.72E−04 −1.07 2.39E−02 (Heyl) 1451501_a_at −1.83 1.69E−02 −2.242.71E−04 −1.66 8.90E−04 −1.12 3.38E−01 (Ghr) 1417714_x_at −8.37 2.49E−02−2.56 4.63E−04 −1.92 1.06E−02 1.42 9.24E−01 (Hba-a1/Hba-a2) 1428361_x_at−8.91 1.93E−02 −2.42 1.08E−03 −1.73 4.27E−02 1.73 4.67E−01(Hba-a1/Hba-a2) 1452590_a_at −1.61 1.07E−02 −1.64 1.22E−02 −1.626.17E−03 1.20 7.36E−01 (Plac9) 1449632_s_at −1.72 1.69E−02 −1.571.12E−02 −1.63 1.80E−04 1.07 5.97E−01 (Fkbp10) 1449280_at 2.07 1.06E−021.55 3.48E−02 1.56 4.35E−02 1.18 2.44E−01 (Esm1) 1418829_a_at 1.792.92E−02 1.71 1.02E−02 1.54 5.43E−03 1.29 9.92E−02 (Eno2)

The expression levels in the stroma from the xenograft models Colo205,B29, B34, B44, PE13, T1 (without estrogen treatment), T1 (with estrogentreatment), C8, and PN8 of selected genes that had been identified inthe microarray analysis as being regulated by treatment with 59R1 (heyl,notch3, rgs5, angpt1, and angpt2) were further analyzed by TaqMan®analysis. The results are shown in FIGS. 12A (heyl), 12B (notch3), 12C(rgs5), 12D (angpt1), and 12E (angpt2).

The results of the TaqMan® analysis confirm that notch3 and rgs5 aredown-regulated in the stroma of each of the various tumor types inresponse to treatment with 59R1 (relative to control) (FIGS. 12B and C).RGS5 is a well-known marker of pericytes and vascular smooth musclecells (Berger et al., 2005, Blood, 105:1094-1101; Lovschall et al.,2007, Int. J. Dev. Biol., 51: 715-721; Cho et al., 2003, FASEB J.,17:440-2). Notch3 has been identified as being coexpressed with RGS5 inpericytes during angiogenesis and playing an important role in theregulation of the fate of pericytes and vascular smooth muscle cells(Lovschall et al., 2007, Int. J. Dev. Biol., 51: 715-721; Domenga etal., 2004, Genes & Dev., 18:2730-2735; Sweeney et al., 2004, FASEB J.,18:1421-3; Morrow et al., 2005, Am. J. Physiol. Cell Physiol.,289:C1188-C1196).

In addition, heyl was also confirmed to be downregulated in the stromaof each of the xenograft models except B34 (FIG. 12A). HeyL belongs tothe Hey family of downstream transcription factors of Notch signaling(Hey1, Hey2, and HeyL). The downregulation of hel by 59R1 suggests thatthe 59R1 antibody directly affects Notch signaling by downregulatingheyl.

Angiopoietin-1 (angpt1) and angiopoietin-2 (angpt2) were also determinedto be down-regulated in the stroma of a number of the breast cancermodels (FIGS. 12D and E). ANGPT1 and 2 (angiopoietin-1 and -2) areligands for the TIE 1 and 2 receptors. TIE receptors, like VEGF, arecrucial signaling molecules in neoangiogenesis processes (Jones et al.,2001, Nature Reviews, 2:257-267).

Notably, however, angpt1 and angpt2 were down-regulated in the stroma ofthe T1 model when estrogen treatment was used (“T1 e”), conditions underwhich treatment with 59R1 was effective against tumor growth, but not inthe stroma of the same model in the absence of estrogen treatment (“T1ne”), conditions under which treatment with 59R1 was ineffective againsttumor growth (see Example 9, above). Thus, the effect of 59R1 on thedown-regulation of angiopoietin-1 and angiopoietin-2 in the stroma ofthe T1 tumor is abrogated in the absence of estrogen treatment. Onepossible explanation of this effect is that in the absence of estrogentreatment, the levels of the growth factors angiopoietin-1 andangiopoietin-2 in the T1 stroma are not sufficiently elevated to providefor measurable decreases in expression levels upon treatment with the59R1 antibody. Estrogen has been shown to have significant effects onthe tumor microenvironment (Banka et al., 2006, Cancer Res.66:3667-3672). One possible explanation of this data is that estrogenleads to a dependence of the tumor on ANGPT2 signaling, which then leadsto sensitivity to 59R11 treatment.

Example 11 Anti-Notch2/3 Antibody 59R1 Significantly Induces Hypoxia inColon and Breast Tumors

Staining for hypoxic regions was performed in Colo-205 colon tumors andPE-13 breast tumors that had been treated either with 59R1 IgG2 antibodyor with IB711 control antibody. The staining was performed as describedin Ridgway et al., 2006, Nature 444:1083-1087. Briefly, to measurehypoxia, pimonidazole-hydrochloride (HypoxyProbe, NPI, Burlington,Mass.), which forms long-lived protein adducts at partial pressure ofoxygen less than approximately 10 mm Hg, was injected intraperitoneallyat 60 mg/kg 1 hr prior to sacrifice. Tumors were then processed forhistological analysis, and tumor sections were stained usinganti-pimonidazole antibody following manufacturer's protocol (NPI).Photographs were taken using a BX51 microscope (Olympus, Center Valley,Pa.).

Viable tumor cells were found to be equally present in IB711 and59R1-treated tumors, as indicated by a relatively uniform and dense DAPIstain (data not shown). The number of CD31-positive cells also remainedunchanged, suggesting that endothelial cell number was not affected by59R1 treatment. In 59R-treated Colo-205 and PE13 tumors, however,hypoxic regions (as detected by anti-pimonidazole antibody) weresignificantly more pronounced than in 1B711 treated tumors (data notshown). AF594-conjugated goat anti-rat F(ab′)₂ was used to detectanti-CD31 antibody and FITC-conjugated goat anti-rabbit antibody wasused to detect anti-pimonidazole antibody.

Example 12 Breast Tumors Comprising Deletions in the PTEN TumorSuppressor Gene are Responsive to Treatment with 59R1

DNA samples were prepared from tumor cells of xenograft breast cancers.Before the DNA isolation, mouse stroma cells in the xenograft tumorswere depleted using magnetic beads conjugated with mouse cell specificantibodies. The purified DNA samples were hybridized to AffymetrixGenome-Wide Human SNP Array 6.0 genechip (Affymetrix, Santa. Clara,Calif.), which has more than 946,000 probes for the detection of copynumber variations (CNVs), according to the manufacturer's instructions.The copy number state changes were estimated by Hidden Markov Model(HMM) and their variations (CNVs) of each sample were obtained by ranksegmentation analysis using Hapmap270 as baseline. Due to the inherentnoise in the array, −0.5 and −1.0 log 2 ratios were used as the cutoffsfor the heterozygous deletion and homozygous deletion under thesignificance threshold <1.0×10⁻⁶ and minimum number of probes persegment=5.

FIG. 13 shows the exon, Affymetrix probe distribution, and the deletionsin the gene of the tumor suppressor phosphatase tensin homolog (PTEN) inchromosome 10. The B29, B34, B37, B40, B51, T2, T3, and T6 breast tumorswere found to have intact PTEN genes in their genomes. The PTEN gene wasdetermined to harbor homozygous deletions in B39 tumor, while B44, PE13,and T1 tumors were determined to have heterozygous deletions of thisgene. As discussed above, 59R1 was determined to have anti-tumorefficacy in each of these four breast tumors comprising homozygous orheterozygous deletions of PTEN. These results suggest that tumors,especially breast tumors, harboring homozygous or heterozygous PTENdeletions may be particularly suitable for treatment with ananti-Notch2/3 antibody such as 59R1.

Example 13 Characterization of 59R5 Antibody

An additional human antibody 59R5 that specifically binds human Notch 2and human Notch 3 was identified. The sequences of the heavy chain andlight chain are provided in SEQ ID NO: 49 and SEQ ID NO:18,respectively. The heavy chain variable region is provided in SEQ IDNO:50 and the light chain variable region is provide SEQ ID NO:13. Theheavy chain CDR3 sequence of 59R5 comprises SIFYTT, SEQ ID NO:51. Theother CDR sequences of 59R5 are identical to 59R1. Biacore analysis of59R1 and 59R5 binding affinities indicated that 59R5 had similar bindingproperties for both Notch2 and Notch3 as 59R1. Both antibodies bindhuman and murine Notch2 and Notch3 receptors with sub-nanomolar affinity(see Table 6).

TABLE 6 IgG Dissociation Constants (K_(D), nM) m-Notch1 h-Notch1m-Notch2 h-Notch2 m-Notch3 h-Notch3 h-Notch4 59R1 >10 >10 0.65 0.05 0.320.19 NB 59R5 >10 >10 0.26 0.05 0.29 0.22 NB

R5 was determined to have similar activity in blocking Notch2 and Notch3signaling as 59R1. Receptor activation was determined inluciferase-based assays. PC3 tumor cells were transiently transfectedwith a human or mouse Notch receptor (human Notch2, murine Notch2, humanNotch3, or murine Notch3) and GFP inducible reporter construct.Transfected cells were incubated with different concentrations of 59R1or 59R5 antibody in the presence of passively immobilized DLL4-Fcprotein. Notch receptor activation was determined by measuringluciferase activity. As shown in FIG. 15A, 59R5 blocked ligand-inducedactivation of human Notch2, murine Notch2, human Notch3 and murineNotch3 receptor signaling at similar levels as 59R1.

The binding epitope of 59R5 was determined. As was described in Example3 for analysis of antibody 59R1, several point mutants were createdwithin full-length Notch1, converting residues within EGF10 to thecorresponding amino acids in human Notch 2. Mutants in fill-length Notchsequences were generated by QuikChange® mutagenesis (Stratagene) andverified by sequencing. HEK 293 cells were transiently transfected withexpression vectors encoding human Notch2, human Notch1, or human Notch1with residues 382-386 mutated to the corresponding human Notch2residues. Cells were also co-transfected with a plasmid encoding greenfluorescent protein (GFP) to mark those cells that received transfectedplasmid. Cells were incubated with 59R1 or 59R5 and fluorescentsecondary antibody and then examined by FACS. 59R1 and 59R5 weredetected by PE-conjugated goat anti-human Fc gamma specific antibody(Jackson Immunochemicals, #109-116-170). As shown in FIG. 15B, 59R5bound to Notch2 and did not bind to Notch1. However, when amino acidscorresponding to Notch2 amino acids 385-389 were substituted intoNotch1, 59R5 was able to bind to the mutated Notch1. This suggested thatat least one or more amino acids necessary for 59R5 binding to humanNotch 2 were positioned within amino acids 385-389 (residues in theboxed hNotch2 sequence shown in FIG. 14A) and suggested that 59R5 bindsthe same epitope as 59R1, or an epitope similar to, or overlapping with,the epitope of 59R1

Example 14 In Vivo Treatment of Tumors Using Notch2/3 Antibody 59R5

In one embodiment, NOD/SCID mice were injected with PE13 breast tumorcells. The mice were treated with anti-Notch2/3 antibody 59R1,anti-Notch2/3 antibody 59R5, or control antibody. Antibodies were dosedat 15 mg/kg once per week in a “preventative” mode where dosing wasinitiated two days after cell injection. FIG. 16A shows that 59R5treatment inhibited tumor growth by greater than 80%, similar to theeffects seen with 59R1.

In another embodiment, NOD/SCID mice were injected with C28 colon tumorcells. The mice were treated with anti-Notch2/3 antibody 59R1,anti-Notch2/3 antibody 59R5 or control antibody. Antibodies were dosedat 15 mg/kg once per week in a “preventative” mode where dosing wasinitiated two days after cell injection. FIG. 16B shows that both 59R1and 59R5 inhibited the growth of C28 colon tumors.

In another embodiment, NOD/SCID mice were injected with Colo205 colontumor cells. The mice were treated with anti-Notch2/3 antibody 59R1,anti-Notch2/3 antibody 59R5 or control antibody. Antibodies were dosedat 15 mg/kg once per week after tumors had been established. FIG. 16Cshows that both 59R1 and 59R5 inhibited the growth of Colo208 colontumors at similar levels.

Example 15 In Vivo Treatment of Tumors Using Notch2/3 Antibody 59R5 inCombination Treatment

In one embodiment, NOD/SCID mice were injected with PN8 pancreatic tumorcells. The tumors were allowed to grow for approximately 33 days untilthey had reached an average tumor volume of 150 mm³. The mice weretreated with gemcitabine at 20 mg/kg once per week for four weeks incombination with control antibody, anti-Notch2/3 antibody 59R1, oranti-Notch2/3 antibody 59R5. As shown in FIG. 17A, antibody 59R5inhibited tumor growth at a similar level as antibody 59R1 and thatcombination treatment prolonged tumor recurrence longer than gemcitabinealone.

In one embodiment, to evaluate the effect of 59R5 on cancer stem cells,a tumor recurrence study was carried out in the PE13 breast tumor model.NOD/SCID mice were injected with PE13 breast tumor cells. The tumorswere allowed to grow for 40 days before treatments were initiated. Themice were treated with taxol at 15 mg/kg twice per week for 5 weeks, incombination with either control antibody or anti-Notch 2/3 antibody59R5. After 5 weeks, the taxol treatments were stopped and the antibodytreatments were continued. 59R5 was observed to significantly delaytumor recurrence after high-dose taxol treatment (FIG. 17B). Theseresults suggest that 59R5 treatment reduces cancer stem cell frequency.

A summary of the in vivo activity of 59R1 and 59R5 as described in thepreceding embodiments is shown in Table 7. Tumor volumes and p valuesfor each experiment are shown relative to the control group. The PE13,C28 and Colo²⁰⁵ studies were carried out as described in Example 14. PN8studies were carried out as described above. For the PN8 experiment, thecontrol is gemcitabine alone and values for 59R1 and 59R5 are thecombinations with gemcitabine. Antibodies were dosed once per week at 15mg/kg for all experiments.

TABLE 7 PE13 C28 Colo205 PN8 Tu- Tu- Tu- Tu- mor p mor p mor p mor p volvalue vol value vol value vol value 59R1 0.25 <0.0001 0.29 <0.0001 0.680.003 0.27 0.026 59R5 0.18 <0.0001 0.38 <0.0001 0.61 0.001 0.11 0.036

Example 16 Regulation of Gene Expression in Tumors after 59R5Treatment

To determine if 59R5 and 59R1 were functioning by the same mechanisms invivo, the expression of key target genes in tumor cells and tumor stromawere examined. Gene expression was assayed by quantitative PCR in PE13tumor cells and stromal cells. Gene expression levels relative to thecontrol antibody treated group are shown in FIG. 18. 59R1 and 59R5regulated the expression of murine HeyL, Notch3, and RGS5 in stromalcells to a similar extent (left panel). The same pattern of regulationwas observed in C28 tumors (data not shown). Thus, the mechanism ofaction previously identified for 59R1 in regulating genes in the tumorstromal critical for function of the tumor vasculature and pericytes wasretained by 59R5. Similarly, 59R5 and 59R1 regulated the expression ofthe human genes ID4, EDNRA, and EGLN3 in tumor cells to the same degree(right panel).

Unlike other members of this gene family, ID4 is generallyunderexpressed in tumors, and ID4 has been shown to be a tumorsuppressor in breast cancer that is frequently silenced by methylation.Loss of expression of ID4 is correlated with a worse prognosis in breastcancer patients (Noetzel et al., 2008, BMC Cancer 8:154). Thus,up-regulation of 1D4 in PE13 breast tumor cells may be part of theanti-tumor mechanism of anti-Notch2/3. EDNRA is the gene encodingendothelin receptor which promotes growth of both endothelial and tumorcells and stimulates metastatic activity of tumor cells (Bagnato andRosano 2008, Int. J. Biochem. Cell. Biol. 40:1443-51). EGLN3 (also knownas HIF-3α) is a hypoxia inducible gene. induction of EGLN3 byanti-Notch2/3 is consistent with disruption of functional vasculature inthe treated tumors. These data indicated that the biological activitiesand mechanism of action of 59R1 and 59R5 were very similar.

Table 8 shows results from a microassay analysis of 59R1 and 59R5treated PE13 tumors. The numbers are mean differential expression valuesfor treated vs. control animals, with 3 animals per group.

TABLE 8 59R1 59R5 Fold pVal Fold pVal Symbol Gene Title −5.10 3.21E−05−3.00 1.10E−03 Foxc2 forkhead box C2 −4.40 1.26E−05 −2.46 7.45E−04 Hey2hairy/enhancer-of-split related with YRPW motif 2 −4.32 8.03E−06 −2.141.00E−04 Rgs5 regulator of G-protein signaling 5 −3.33 5.59E−04 −2.793.18E−03 Hey1 hairy/enhancer-of-split related with YRPW motif-like −2.714.80E−04 −2.90 1.02E−04 Rgs4 regulator of G-protein signaling 4 −2.102.17E−04 −1.86 4.33E−05 Notch3 Notch gene homolog 3 (Drosophila) −1.923.16E−02 −2.35 3.43E−03 Mmp9 matrix metallopeptidase 9 2.36 3.06E−024.97 3.35E−02 Pdcd1lg2 programmed cell death 1 ligand 2 7.42 6.25E−072.80 2.07E−03 Gzma granzyme A

Microarray analysis reveals that 59R5 significantly inhibited the Notchpathway (p<0.01) as measured by gene expression (e.g., Foxc2, Hey2,Heyl, Notch3). These results were comparable to 59R1. Foxc2 is adown-stream target of the Hedgehog pathway and is involved in celldifferentiation. Additional genes involved in apoptosis (e.g., granzymeA) and tumor-associated tissue remodeling (MMP-9) were also similarlyexpressed between 59R1 and 59R5. These data suggested that thebiological activities and mechanism of action of 59R1 and 59R5 are verysimilar.

Example 17 Production of additional Notch2 and/or Notch3 AntibodiesAntigen Production

In certain embodiments, recombinant polypeptide fragments of the humanNotch2 or human Notch3 extracellular domain are generated as antigensfor antibody production. For example, standard recombinant DNAtechnology can be used to isolate a polynucleotide encoding amino acids1-493 of Notch2 (SEQ ID NO: 33), encompassing EGF 1-12. Thispolynucleotide can be ligated in-frame N-terminal to either a humanFc-tag or histidine-tag and cloned into a transfer plasmid vector forbaculovirus mediated expression in insect cells. Standard transfection,infection, and cell culture protocols can be used to produce recombinantinsect cells expressing the corresponding Notch2 polypeptide (SEQ ID NO:34) (O'Reilly et al., 1994, Baculovirus expression vectors: A LaboratoryManual, Oxford: Oxford University Press).

Cleavage of the endogenous signal sequence of human Notch2 wasapproximated using cleavage prediction software SignalP 3.0, however theactual in vivo cleavage point can differ by a couple of amino acidseither direction. The predicted cleavage of Notch2 is between aminoacids 1 and 26, thus Notch2 antigen protein comprises approximatelyamino acid 27 through amino acid 493. Antigen protein can be purifiedfrom insect cell conditioned medium using Protein A and Ni⁺⁺-chelateaffinity chromatography. Purified antigen protein is then dialyzedagainst PBS (pH=7), concentrated to approximately 1 mg/ml, and sterilefiltered in preparation for immunization.

Immunization

Mice can be immunized with purified Notch2 or Notch3 antigen proteinusing standard techniques. Blood from individual mice can be screenedapproximately 70 days after initial immunization for antigen recognitionusing ELISA and FACS analysis (described in detail below). The animalswith the highest antibody titers are then selected for final antigenboost after which spleen cells are isolated for hybridoma production.Hybridoma cells are plated at 1 cell per well in 96 well plates, and thesupernatant from each well screened by ELISA and FACS analysis againstantigen protein. Several hybridomas with high antibody titer areselected and scaled up in static flask culture. Antibodies are purifiedfrom the hybridoma supernatant using protein A or protein G agarosechromatography and antibodies are tested by FACS as described below.

FACS Analysis

To select monoclonal antibodies produced by hybridomas, clones thatrecognize native cell-surface Notch2 (and/or Notch3) protein, FACsanalysis is used. HEK293 cells are co-transfected with expressionvectors encoding a full-length cDNA clone of Notch2 and the transfectionmarker GFP. Twenty-four to forty-eight hours post-transfection, cellsare collected in suspension and incubated on ice with anti-Notch2 (oranti-Notch3 or anti-Notch2/3) antibodies or control IgG to detectbackground antibody binding. The cells are washed and primary antibodiesdetected with anti-mouse secondary antibodies conjugated to afluorescent chromophore. Labeled cells are then sorted by FACS toidentify anti-Notch2, anti-Notch3, or anti-Notch2/3 antibodies thatspecifically recognize cell surface expression of native cell-surfaceNotch2 and/or Notch3 protein.

Chimeric Antibodies

After monoclonal antibodies that specifically recognize a non-ligandbinding domain of a Notch receptor are identified, these antibodies aremodified to overcome the human anti-mouse antibody (HAMA) immuneresponse when rodent antibodies are used as therapeutics agents. Thevariable regions of the heavy-chain and light-chain of the selectedmonoclonal antibody are isolated by RT-PCR from hybridoma cells andligated in-frame to human IgG1 heavy-chain and kappa light chainconstant regions, respectively, in mammalian expression vectors.Alternatively a human Ig expression vector such as TCAE 5.3 is used thatcontains the human IgG1 heavy-chain and kappa light-chain constantregion genes on the same plasmid (Preston et al., 1998, Infection &Immunity 66:4137-42). Expression vectors encoding chimeric heavy- andlight-chains are then co-transfected into Chinese hamster ovary (CHO)cells for chimeric antibody production. immunoreactivity and affinity ofchimeric antibodies are compared to parental murine antibodies by ELISAand FACS.

Humanized Antibodies

As chimeric antibody therapeutics are still frequently antigenic,producing a human anti-chimeric antibody (HACA) immune response,chimeric antibodies against a Notch2 or Notch3 receptor can requirefurther humanization. To generate humanized antibodies the three shorthypervariable sequences, or complementary determining regions (CDRs), ofthe chimeric antibody heavy- and light-chain variable domains describedabove are engineered using recombinant DNA technology into the variabledomain framework of a human heavy- and light-chain sequences,respectively, and then cloned into a mammalian expression vector forexpression in CHO cells. The immunoreactivity and affinity of thehumanized antibodies are compared to parental chimeric antibodies byELISA and FACS. Additionally, site-directed or high-density mutagenesisof the variable region can be used to optimize specificity, affinity,etc. of the humanized antibody.

Example 18 Additional In Vitro Assays to Evaluate Antibodies Against aNotch Receptor

This example describes methods for in vitro assays to test the activityof antibodies generated against a Notch2 and/or Notch3 receptor on cellproliferation and cytotoxicity.

Proliferation Assay

Antibodies against Notch2 and/or Notch3 are tested for their effect ontumor cell growth in vitro using a BrdU based assay. Freshlydissociated, Lin-depleted breast tumor cells are cultured in low oxygenfor between 2-5 days. Cells are then cultured at 20,000 cells/well with2.5 μg/mL or 5.0 μg/mL anti-Notch antibody, control non-specific murineIgG, or no antibody for three days followed by 18 hours BrdU labeling.All experiments are performed with multiple replicates. The ability ofanti-Notch antibodies to inhibit cell proliferation compared to controlantibodies is then determined.

Complement-Dependent Cytotoxicity Assay

Cancer cell lines expressing a Notch2 receptor and/or a Notch3 receptoror, alternatively, cancer stem cells isolated from a patients samplepassaged as a xenograft in immunocompromised mice are used to measurecomplement dependent cytotoxicity (CDC) mediated by an antibody againsta Notch 2 and/or Notch3 receptor. Cells are suspended in 200 μl RPMI1640 culture medium supplemented with antibiotics and 5% FBS at 106cells/ml. Suspended cells are then mixed with 200 μl serum orheat-inactivated serum with antibodies against a Notch2 and/or Notch3receptor or control antibodies in triplicate. Cell mixtures areincubated for 1 to 4 hours at 37° C. in 5% CO₂. Treated cells are thencollected, resuspended in 100 μl FITC-labeled annexin V diluted inculture medium and incubated at room temperature for 10 min. One hundredμl of a propidium iodide solution (25 μg/ml) diluted in HBSS is addedand incubated for 5 min at room temperature. Cells are collected,resuspended in culture medium and analyzed by flow cytometry. Flowcytometry of FITC stained cells provides total cell counts, andpropidium iodide uptake by dead cells as a percentage of total cellnumbers is used to measure cell death in the presence of serum andantibodies against a Notch2 and/or Notch3 receptor compared toheat-inactivated serum and control antibodies. The ability ofanti-Notch2/3 antibodies to mediated complement-dependent cytotoxicityis thus determined.

Antibody-Dependent Cellular Cytotoxicity Assay

Cancer cell lines expressing a Notch2 receptor and/or a Notch3 receptoror, alternatively, cancer stem cells isolated from a patients samplepassaged as a xenograft in immunocompromised mice (described in detailbelow) are used to measure antibody dependent cellular cytotoxicity(ADCC) mediated by an antibody against a Notch2 and/or Notch3 receptor.Cells are suspended in 200 μl phenol red-free RPMI 1640 culture mediumsupplemented with antibiotics and 5% FBS at 106 cells/ml. Peripheralblood mononuclear cells (PBMCs) are isolated from heparinized peripheralblood by Ficoll-Paque density gradient centrifugation for use aseffector cells. Target cells (T) are then mixed with PBMC effector cells(E) at E/T ratios of 25:1, 10:1 and 5:1 in 96-well plates in thepresence of a Notch2 or Notch3 receptor or control antibodies. Controlsinclude incubation of target cells alone and effector cells alone in thepresence of antibody. Cell mixtures are incubated for 1 to 6 hours at37° C. in 5% CO₂. Released lactate dehydrogenase (LDH), a stablecytosolic enzyme released upon cell lysis, is then measured by acolorimetric assay (e.g., CytoTox96 Non-radioactive Cytotoxicity Assay;Promega; Madison, Wis.). Absorbance data at 490 nm are collected with astandard 96-well plate reader and background corrected. The percentageof specific cytotoxicity is calculated according to the formula: %cytotoxicity=100×(experimental LDH release−effector spontaneous LDHrelease−target spontaneous LDH release)/(target maximal LDHrelease−target spontaneous LDH release). The ability of antibodiesagainst a Notch2 and/or Notch3 receptor to mediated antibody dependentcellular cytotoxicity is thus determined.

Example 19 Production of Antibodies Against EGF 10 (or Equivalent EGF)of Notch Receptors

Identification of an antibody that specifically binds the tenth EGFrepeat of Notch2 and the corresponding EGF repeat of Notch3 (the ninthEGF repeat) that reduces tumor growth in animals suggests the importanceof the non-ligand binding domain, and the tenth EGF repeat (or itsequivalent) in particular, for effective cancer therapies. To target theEGF repeat 10 (or equivalent EGF) in Notch receptor family members,antibodies against EGF10 of Notch1, Notch2, or Notch4 or against EGF9 ofNotch3 are produced and analyzed. Specifically, mice are immunized withantigens comprising the tenth EGF repeat of Notch1 (SEQ ID NO:35);Notch2 (SEQ ID NO:36), or Notch4 (SEQ ID NO:38) or the ninth EGF repeatof Notch3 (SEQ ID NO:37). Antibodies that recognize specific Notchreceptors as well as antibodies that recognize different combinations ofthe four Notch receptors are identified using FACS analysis of HEK 293cells transfected with each Notch receptor as described in detail above.Antibodies that recognize the tenth EGF repeat (or equivalent EGF) oftwo Notch receptor family members are envisioned (e.g. antibodies thatrecognize the Notch1 EGF 10 and Notch2 EGF10; Notch1 EGF10 and Notch3EGF9; Notch1 EGF10 and Notch4 EGF10; Notch2 EGF10 and Notch3 EGF9;Notch2 EGF10 and Notch4 EGF10; or Notch3 EGF9 and Notch4 EGF10).Antibodies that recognize the tenth EGF repeat (or equivalent EGF) ofthree Notch receptor family members are likewise contemplated (e.g.,antibodies that recognize the Notch1 EGF10, Notch2 EGF10, and Notch3EGF9; Notch1 EGF10, Notch2 EGF10, and Notch4 EGF10; or Notch2 EGF10,Notch3 EGF9, and Notch4 EGF10). And antibodies that recognize the tenthEGF repeat (or equivalent EGF) of four Notch receptor family members areenvisioned (e.g. antibodies that recognize the Notch1 EGF10, Notch2EGF10, Notch3 EGF9 and Notch4 EGF10).

Example 20 Treatment of Human Cancer Using Anti-Notch ReceptorAntibodies

This example describes methods for treating cancer using antibodiesagainst a Notch receptor to target tumors comprising cancer stem cellsand/or tumor cells in which Notch receptor expression has been detected.

The presence of cancer stem cell marker expression can first bedetermined from a tumor biopsy. Tumor cells from a biopsy from a patientdiagnosed with cancer are removed under sterile conditions. In someembodiments, the tissue biopsy is fresh-frozen in liquid nitrogen,embedded in O.C.T., and cut on a cryostat as 10 μm sections onto glassslides. Alternatively the tissue biopsy is formalin-fixed,paraffin-embedded, and cut on a microtome as 10 μm section onto glassslides. Sections are incubated with antibodies against a Notch receptorto detect protein expression. Additionally, the presence of cancer stemcells can be determined. Tissue biopsy samples are cut up into smallpieces, minced completely using sterile blades, and cells subject toenzymatic digestion and mechanical disruption to obtain a single cellsuspension. Dissociated tumor cells are then incubated with anti-ESA,-CD44, -CD24, and -Lin, antibodies to detect cancer stem cells, and thepresence of ESA+, CD44+, CD24−/low, Lin− tumor stem cells is determinedby flow cytometry as described in detail above.

Cancer patients whose tumors are diagnosed as expressing a Notchreceptor are treated with anti-Notch receptor antibodies. Humanized orhuman monoclonal anti-Notch receptor antibodies generated as describedabove are purified and formulated with a suitable pharmaceutical carrierin PBS for injection. Patients are treated with the Notch antibodiesonce a week for at least 10 weeks, but in certain cases once a week forat least about 14 weeks. Each administration of the antibody should be apharmaceutically effective dose about 2 to about 100 mg/ml and incertain cases between about 5 to about 40 mg/ml. The antibody can beadministered prior to, concurrently with, or after standard radiotherapyregimens or chemotherapy regimens using one or more chemotherapeuticagent, such as paclitaxel, gemcitabine, irinotecan, oxaliplatin,fluorouracil, leucovorin, or streptozocin. Patients are monitored todetermine whether such treatment has resulted in an anti-tumor response,for example, based on tumor regression, reduction in the incidences ofnew tumors, lower tumor antigen expression, decreased numbers of cancerstem cells, or other means of evaluating disease prognosis.

Example 21 Production of Antibodies Against Notch1, Notch2, Notch3,and/or Notch4 EGF Repeat 4

To target the EGF repeat 4 in Notch receptor family members, antibodiesagainst Notch1, Notch2, Notch3, and/or NOTCH4 EGF repeat 4 are producedand analyzed. Specifically, mice are immunized with antigens comprisingthe fourth EGF repeat of Notch1 (SEQ ID NO:41), Notch2 (SEQ ID NO:42),Notch3 (SEQ ID NO: 43), or Notch4 (SEQ ID NO:44). Antibodies thatrecognize specific Notch receptors as well as antibodies that recognizedifferent combinations of the four Notch receptors are identified usingFACS analysis of HEK 293 cells transfected with each Notch receptor asdescribed in detail above. Antibodies that recognize the fourth EGFrepeat of two Notch receptor family members are envisioned (e.g.antibodies that recognize the fourth EGF repeat of Notch1 and Notch2;Notch1 and Notch3; Notch1 and Notch4; Notch2 and Notch3; Notch2 andNotch4; or Notch3 and Notch4). Antibodies that recognize the fourth EGFrepeat of three Notch receptor family members are envisioned (e.g.antibodies that recognize the fourth EGF repeat of Notch1, Notch2, andNotch3; Notch1, Notch2, and Notch4; or Notch2, Notch3, and Notch4). Andantibodies that recognize the fourth EGF repeat of four Notch receptorfamily members are envisioned (e.g. antibodies that recognize the fourthEGF repeat of Notch1, Notch2, Notch3 and Notch4).

A description of the exemplary production and characterization of amonoclonal antibody, 13M57, that binds EGF4 of Notch1 can be found inU.S. Patent Application Publication No. 2008/0131434, which isincorporated by reference herein in its entirety.

Example 22 Additional Gene Expression Assays in Tumor Cells Treated with59R1

Changes in gene expression in response to 59R1 treatment in tumor cellsin xenograft models were identified.

Several pathways/gene sets that are regulated by antibody 59R1 in tumorcells were identified (Table 9) using Gene Set Enrichment Analysis(Mootha et al., 2003, Nature Genetics 34:267-73; Subramanian et al.,2005, Proc. Natl. Acad. Sci. USA 102:15545-50) in the breast tumors T1,PE13, and B51. Notably, cell cycle gene pathways, myc-activating genesand several stem cell gene sets are down-regulated by 59R1 in thisanalysis. cMyc has been shown to be a direct target of the Notch pathway(Weng et al., 2006, Genes Dev. 20:2096-109). The stem cell gene setsdown-regulated by 59R1 were derived from a molecular signature derivedfrom five distinct populations: human fetal hematopoietic stem cells(HCS), murine fetal and adult HSCs, neural stem cells (NSC), andembryonic stem cells (ESC) (Ivanova et al., 2002, Science 298:601-604),and also a recently described core ESC gene set (Ben-Porath et al.,2008, Nature Genetics 40:499-507) and an ESC self-renewal gene set whosedown-regulation causes differentiation (Hu et al., 2009, Genes Dev.23:837-48).

TABLE 9 Name Size FDR Description NGUYEN KERATO UP 27 0.0774 Genesconcomitantly modulated by activated NOTCH Notch 1 in mouse and humanprimary keratinocytes- Up CELLCYCLEPATHWAY 22 0.0798 Cyclins interactwith cyclin-dependent kinases to form active kinase complexes thatregulate progression thr YU CMYC UP 28 0.0884 Myc-activated genes HSCSTHSC FETAL 27 0.0885 Up-regulated in mouse short-term functionalhematopoietic stem cells from fetal liver (ST-HSC Shared) HSC STHSCSHARED 27 0.0907 Up-regulated in mouse short-term functionalhematopoietic stem cells from both adult bone marrow an WEINBERG ESCEXP2 30 0.1001 40 genes specifically overexpressed in hES cellsaccording to Meta-analysis of 8 profiling studies (Natu ESC SELF RENEWAL30 0.1087 Genes identified by a genome-wide RNAI screen, whosedownregulation caused mESC differentiation BRENTANI REPAIR 33 0.1122Cancer related genes involved in DNA repair FDR < 15%

Example 23 Reduction of Cancer Stem Cell Frequency by Notch2/3Antibodies

Using a similar experimental study as described in Example 8, ananalysis of cancer stem cell frequency by limiting dilution analysis wascarried out in PE13 breast cancer cells. Animals bearing PE13 breasttumors were treated with control antibody, taxol plus control antibody,59R1, or taxol plus 59R1 for three weeks. Tumors were harvested afterthree weeks, and CSC frequency in the treated tumors was analyzed.Serial titrations of human cells from each the four treatment groupswere transplanted into a new set of mice (n=10 per cell dose). Tumorgrowth rate after 75 days of growth (FIG. 19A) was used to calculate theCSC frequency using the L-calc program (Stem Cell Technologies, Inc.).The control antibody treated tumors were determined to have a tumorinitiating cell frequency of 1:74. Treatment with taxol alone increasedthe CSC frequency to 1:30. In contrast, treatment with 59R1 decreasedCSC frequency to 1:179 and the combination of 59R1 plus taxol decreasedCSC frequency to 1:319 (FIG. 19B). A single asterisk indicates astatistically significant difference (p<0.05) vs. the control antibodytreated group and a double asterisk indicates a significant differencevs. the taxol and control antibody treated group. This experimentindicated that 59R1 treatment of PE13 breast tumors reduced CSCfrequency as a single agent and more dramatically, in combination withtaxol treatment. In contrast, treatment with taxol alone, whileeffective at reducing tumor volume, increased the CSC frequency oftreated tumors indicating that tumor initiating cells are preferentiallyresistant to the effects of this chemotherapeutic agent.

In addition to investigating the effects of 59R1 in tumors and theeffect on CSC frequency, gene changes were studied following 59R1treatment in combination with taxol. The experiment was performed inPE13 breast tumors where a decrease in CSC frequency after treatmentwith 59R1 alone or 59R1 plus taxol treatment had previously beenobserved (and described herein). Microarray analysis was performed ontumors from the same experiment where limiting dilution analyses of PE13were carried out for CSC quantification (FIG. 19). Animals bearing PE13breast tumors were treated three weeks with 59R1 plus taxol, control andtaxol prior to harvesting for microarray analysis. Mean differentialexpression values for taxol vs. control and 59R1 plus taxol vs. taxoltreated animals (3 animals per group) were calculated. Strikingly, inthe gene expression microarray data, it was found that 59R1 incombination with taxol affected apoptosis, hypoxia, differentiation, andstem-cell related genes in the opposite fold direction than the genechanges observed following with taxol alone (Table 10) consistent withthe effects of these compounds on the CSC frequency.

TABLE 10 Taxol vs. control 59R1 Taxol vs. Taxol Fold pval Fold pvalSymbol Name 10.2 6.8E−03 −4.3 2.3E−01 BMPR1B bone morphogenetic proteinreceptor, type IB −2.1 4.2E−05 1.8 6.9E−05 BNIP3 BCL2/adenovirus E1B19kDa interacting protein 3 −21.1 1.0E−02 11.9 3.9E−04 EGLN3 egl ninehomolog 3 13.4 5.9E−05 −1.8 1.2E−01 HSPB6 heat shock protein,alpha-crystallin-related, B6 2.2 1.5E−02 −2.5 1.9E−03 ITGAM integrin,alpha M 4.6 3.6E−03 −4.4 5.2E−03 LHX8 LIM homeobox 8 −9.0 2.0E−06 6.41.8E−05 NDRG1 N-myc downstream regulated gene 1 6.4 6.9E−06 −2.2 7.4E−03RARRES1 retinoic acid receptor responder 1 2.6 3.5E−04 −1.7 1.1E−03RARRES3 retinoic acid receptor responder 3 4.8 3.9E−05 −2.2 1.6E−02 RBP2retinol binding protein 2, cellular 10.6 1.3E−10 −1.5 5.9E−02 XAF1 XIAPassociated factor 1

The apoptosis-related genes regulated in this dataset include BNIP3,NDRG1 HSPB6, and XAF1. BNIP3 (Bcl-2/E1B 19 kDa interacting protein) is apro-apoptotic member of the Bcl-2 family that is expressed in hypoxicregions of tumors (Kothari et al., 2003, Oncogene 22:4734-44). BNIP3 isdown-regulated by taxol alone and up-regulated by the combinationtherapy, suggesting that 59R1 plus taxol may promote apoptosis.Consistent with this idea is the observation that HSPB6 isdown-regulated in taxol treated tumors; HSPB6 over-expression mayprotect against apoptosis in some biological systems (Fan et al., 2005,Trends Cardiovasc. Med. 15:138-41). NDRG1 (N-myc downstream regulatedgene1), which is up-regulated in the combination treatment, is necessaryfor p53-dependent apoptosis (Stein et al., 2004, J. Biol. Chem.279:48930-40). Interestingly, NDRG1 is also a putative suppressor ofcolorectal cancer metastases. Its increased expression is associatedwith improved survival in prostate and breast cancer (Shah et al., 2005,Clin. Cancer Res. 11:3296-302). Additionally, NDRG1 is involved inpromoting differentiation. The expression of NDRG1 has been shown to behighly expressed in well-differentiated pancreatic cancer cells, and notexpressed in the less differentiated tumor cells (Angst et al., 2006,Br. J. Cancer 95:307-13). It was also observed that other stemcell-related genes such BMPR1B and homeobox containing gene, LHX8, wereup-regulated by taxol alone, and then down-regulated with 59R1 treatmentin combination with taxol.

Several genes involved in the metabolism of retinoids (RARRES1, RARRES3,RBP2), which are similar functionally to the putative stem cell marker,ALDH1a1, were up-regulated by taxol, and then down-regulated with taxolplus 59R1 treatment. Retinoic acid signaling has been shown to be linkedto cellular differentiation (Appel and Eisen, 2003, Neuron 40:461-4).Taken together, these data. show that 59R1 has significant effects ongene expression in PE13 breast tumor cells and may begin to elucidatesome of the mechanisms that underlie the observed decrease in cancerstem cell frequency in this tumor following treatment with 59R1 andtaxol combination therapy.

In another embodiment, a PN4 pancreatic tumor model was used to test forreduction in cancer stem cell frequency after treatment with 59R1. PN4pancreatic tumors were treated with control antibody, anti-Notch2/359R1, gemcitabine, or a combination of 59R1 and gemcitabine for a periodof three weeks. Antibodies were dosed at 10 mg/kg, twice per week andgemcitabine was dosed at 50 mg/kg, twice per week. Tumors from eachgroup were harvested and processed to obtain single cell suspensions.The human tumors cells in the xenograft were isolated and counted. Atitration of cells (30, 90 or 210 cells) were re-injected into NOD-SCIDmice (n=10 per group). Tumor growth was assayed on day 84 and tumorinitiating cell frequency was calculated from the tumor take rate. Thecontrol antibody treated tumors were determined to have a tumorinitiating cell frequency of 1:137. Treatment with gemcitabine aloneincreased the CSC frequency to 1:61. In contrast, treatment with 59R1decreased CSC frequency to 1:281 and the combination of 59R1 plusgemcitabine decreased CSC frequency to 1:675 (FIG. 19C). A singleasterisk indicates a statistically significant difference (p<0.05) vs.the control antibody treated group and a double asterisk indicates asignificant difference vs. the gemcitabine and control antibody treatedgroup.

In another embodiment, a PE13 breast tumor model was used to test forreduction in cancer stem cell frequency after treatment with 59R5. PE13breast tumors were treated with control antibody, anti-Notch2/3 59R5,taxol, or a combination of 59R5 and taxol for a period of three weeks.Antibodies were dosed at 20 mg/kg, once per week and taxol was dosed at15 mg/kg, twice per week. Tumors from each group were harvested andprocessed to obtain single cell suspensions. The human tumors cells inthe xenograft were isolated and counted. A titration of cells (50, 150or 450 cells) were re-injected into NOD-SCID mice (n=10 per group).Tumor growth was assayed on day 39 and tumor initiating cell frequencywas calculated from the tumor take rate. The control antibody treatedtumors were determined to have a tumor initiating cell frequency of1:70. Treatment with taxol alone increased the CSC frequency to 1:30. Incontrast, treatment with 59R5 decreased CSC frequency to 1:202 and thecombination of 59R5 plus taxol decreased CSC frequency to 1:382 (FIG.19D). A single asterisk indicates a statistically significant difference(p<0.05) vs. the control antibody treated group and a double asteriskindicates a significant difference vs. the taxol and control antibodytreated group.

As observed in other experiments, these results indicated that 59R1treatment of PN4 pancreatic tumors and 59R5 treatment of PE13 breasttumors reduced CSC frequency as a single agent and more dramatically, incombination with gemcitabine or taxol treatment, respectively. Incontrast, treatment with taxol or gemcitabine alone, while effective atreducing tumor volume, increased the CSC frequency of treated tumorsindicating that tumor initiating cells are preferentially resistant tothe effects of these chemotherapeutic agents.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those in the relevant fields areintended to be within the scope of the following claims.

1-89. (canceled)
 90. An isolated monoclonal antibody that specificallybinds epidermal growth factor (EGF) repeat 10 of human NOTCH2 and EGFrepeat 9 of human NOTCH3.
 91. The antibody of claim 90, which binds atleast part of the sequence HKGAL (SEQ ID NO:28) within the EGF repeat 10of human NOTCH2.
 92. The antibody of claim 90, which binds at least partof the sequence HEDAI (SEQ ID NO:29) within the EGF repeat 9 of humanNOTCH3.
 93. The antibody of claim 91, which binds at least part of thesequence HEDAI (SEQ ID NO:29) within the EGF repeat 9 of human NOTCH3.94. The antibody of claim 90, which binds human NOTCH2 with a K_(D) ofabout 1 nM or less.
 95. The antibody of claim 94, which binds humanNOTCH3 with a K_(D) of about 1 nM or less.
 96. The antibody of claim 90,which binds human NOTCH3 with a K_(D) of about 1 nM or less.
 97. Theantibody of claim 90, which is a recombinant antibody, a chimericantibody, a humanized antibody, a human antibody, a bispecific antibody,a monospecific antibody, an antibody fragment, an IgG1 antibody, or anIgG2 antibody.
 98. The antibody of claim 97, which is a human antibody.99. The antibody of claim 97, which is a humanized antibody.
 100. Theantibody of claim 97, which is an IgG1 antibody.
 101. The antibody ofclaim 97, which is an IgG2 antibody.
 102. The antibody of claim 90,which is an antagonist of human NOTCH2 and/or human NOTCH3.
 103. Theantibody of claim 90, which inhibits binding of a ligand to human NOTCH2and/or human NOTCH3.
 104. The antibody of claim 90, which inhibits humanNOTCH2 and/or human NOTCH3 signaling.
 105. The antibody of claim 90,which inhibits tumor growth.
 106. An isolated antibody that competes forspecific binding to human NOTCH2 and/or human NOTCH3 with the antibodyof claim 90.